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ORIGINAL COMMUNICATION

Acute effect of high-calcium milk with or without

additional magnesium, or calcium phosphate onparathyroid hormone and biochemical markers ofbone resorption

JH Green1*, C Booth1 and R Bunning1

1Milk and Health Research Centre, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, NewZealand

Objective: To assess whether there are any differences in the postprandial physiological responses to apple drink (control),calcium phosphate (tricalcium phosphate, TCP) and high-calcium skim milk (HCSM) with or without additional magnesium inpostmenopausal women.Design: Randomized, controlled, cross-over. Measurements after overnight fast before each drink, and subsequently every hourfor 8 h postprandially.Setting: Human Nutrition Studies Laboratory, Milk and Health Research Centre, Massey University, Palmerston North, NewZealand.Subjects: Twenty-one healthy postmenopausal women.Intervention: Four drinks, each 400 ml. (1) Apple drink (25% fruit juice). (2) TCP dispersed in water containing 1200mg Ca. (3)HCSM containing 1200 mg Ca and 65.5 mg Mg. (4) HCSM containing 1200 mg Ca and 172 mg Mg.Results: There was no difference in baseline serum calcium, PTH or C-telopeptide levels between drinks. There were no overalldifferences in serum calcium after apple or after either milk, but after TCP serum calcium increased from a baseline value of2.120.08 to a mean peak of 2.210.12 mmol=l (s.d.) (P0.0001) after 2 h. There were no significant differences in serumPTH after either apple or HCSMMg. In contrast, after TCP, serum PTH decreased from 2.760.69 to a mean nadir of2.230.65pmol=l (P0.0001) after 1 h, and after HCSM, it decreased from 2.710.78 to a mean nadir of 2.510.87 pmol=l(P0.007) after 2 h. Serum C-telopeptides decreased after each drink, reaching nadirs after 5 h. At this time the serum values foreach of the high calcium drinks were not different from each other, but were significantly less than for apple ( P0.001 for each),being 0.220.09ng=ml for apple, 0.150.08 for TCP, 0.140.07 for HCSM and 0.16 0.07 for HCSMMg.Conclusion: Despite differences in serum calcium and PTH responses to the three high-calcium drinks that we tested, there wasno distinguishable difference in serum C-telopeptides between high calcium drinks.Sponsorship: New Zealand Milk.European Journal of Clinical Nutrition (2003) 57, 61 68. doi:10.1038=sj.ejcn.1601501

Keywords: calcium; PTH; milk; bone turnover; pyridinium crosslinks; postmenopausal women

IntroductionIt is well established that an acute oral dose of calcium

rapidly suppresses bone resorption (Goddard et al, 1986;

Reid et al, 1986; Woo et al, 1991; Reginster et al, 1993;

Horowitz et al, 1994; McKane et al, 1996; Rubinacci et al,

1996; Yang et al, 1994). However, it is not known whether

high-calcium milk is as effective in this respect. One might

expect that the additional micro- and macronutrients in

milk compared with calcium salts may slow down calcium

absorption and lead to a less marked impact on serum

*Correspondence: JH Green, Milk and Health Research Centre, Institute of

Food, Nutrition and Human Health, Massey University, Private Bag 11222,

Palmerston North, New Zealand.

E-mail: J.H.Green@massey.ac.nz

Guarantor: Dr JH Green.

Contributors: JHG was responsible for the study design, data analysis

and preparation of the paper. CB was responsible for recruiting

subjects, laboratory organisation and data collection. KB assisted with

sample processing and data entry.

Received 2 August 2001; revised 26 March 2002;

accepted 28 March 2002

European Journal of Clinical Nutrition (2003) 57, 6168 2003 Nature Publishing Group All rights reserved 09543007/03 $25.00

www.nature.com/ejcn

European Journal of Clinical Nutrition (2003) 57, 6168 2003 Nature Publishing Group All rights reserved 09543007/03 $25.00

www.nature.com/ejcn

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calcium, parathyroid hormone (PTH) and markers of bone

resorption.

Milk is a good source of magnesium as well as calcium. It

has been known for many years that magnesium (Mg)

deficiency leads to an increase in serum PTH, which pro-

motes calcium (Ca) release from bone (Rude et al, 1976).

More recently Mg deficiency for just 3 weeks in young,

growing rats has been shown to lead to decreased bone

turnover that could impair bone growth and development

(Creedon et al, 1999). Correcting deficiencies in Mg intake is

therefore likely to be important for bone health. However,

there are also advocates for the use of Mg supplementation

for bone health even when Mg status is known not to be

compromised (Abraham & Grewal 1990; Driessens et al,

1990; Stendig-Lindberg et al, 1993). This recommendation

is supported by observations of increased bone mineral

density (Abraham & Grewal 1990; Stendig-Lindberg et al,

1993), as well as relief of backpain and movement restric-

tions by Mg supplementation in osteoporotic patients (Dries-

sens et al, 1990). Also, a number of dietary studies have

consistently suggested that bone mineral density is greater inpeople with high intakes of Mg and potassium (Freudenheim

et al, 1986; Angus et al, 1988; Tranquilli et al, 1994; New et al,

1997; Tucker et al, 1999). There is limited research into the

impact of supplementary Mg on biochemical markers of

bone turnover. Serum PTH and biochemical markers of

bone turnover have been shown to be transiently reduced

during the first 5 10 days of 30 days of Mg supplementation

in young men (Dimai et al, 1998). However, Mg supplemen-

tation for longer than 2 weeks does not lead to significant

changes in bone resorption markers or serum PTH (Dimai

et al, 1998; Doyle et al, 1999; Basso et al, 2000). We have

previously observed decreases in bone resorption in response

to 4 weeks of supplementary Mg-enriched high Ca milk thatwere greater than those seen for high Ca milk without

additional Mg (Green et al, 2000), but that study did not

include any measurements of serum PTH, Ca or Mg.

The present study was designed to assess whether there

are any differences in the postprandial physiological

responses to apple drink (control), calcium phosphate (tri-

calcium phosphate, TCP) and high-calcium skim milk

(HCSM) with or without additional Mg in postmenopausal

women.

Methods

The volunteers were 21 healthy women who were at least 5 ypostmenopausal, and had not taken any mineral supple-

ments or pharmaceutical therapy known to affect bone

metabolism for the 3 y before the study. All the subjects

gave written, informed consent to take part in the study,

which was approved both by the Manawatu-Whanganui

Ethics Committee and the Massey University Human

Ethics Committtee. Body weight was measured using a

beam balance (Detecto, Cardinal Scale Manufacturing, MO,

USA) to the nearest 0.2 kg and standing height was measured

using a stadiometer (Institute of Fundamental Sciences,

Engineering Services Workshop, Massey University, Palmer-

ston North, New Zealand) to the nearest 0.1 cm. Waist and

hip circumferences were measured to the nearest 0.1cm

using a non-stretch measuring tape. Lean body mass was

measured by total body bioelectrical impedence (Biody-

namics Model 310, Seattle, WA, USA) after at least 3 h with-

out food or drink. The maximal handgrip of the non-

dominant arm was measured to the nearest 0.5kg using a

conventional hydraulic handgrip dynamometer (JamarTM,

Sammons Preston Inc, Bolingbrook, IL, USA). Food intake

was assessed on each laboratory visit by 24 h food intake

recalls. The dietary composition of macro- and micronutri-

ent content food intakes was subsequently taken from the

New Zealand Food Composition Table, which we accessed

using nutrient analysis software (FOODworks v.2, Xyris Soft-

ware (Australia) Pty Ltd, Highgate Hill, QLD, Australia).

The volunteers attended the laboratory on four different

days, each at least 1 week apart. They consumed 400 ml of a

different drink, in random order, on each day. An apple drink

containing 25% fruit juice (No FrillsTM, Franklins Ltd, Chul-lora, NSW, Australia) served as the control drink. The apple

drink provided 0.07g Ca, 0.05mg Mg and

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was kept cold until it was spun at 3000 rpm for 10 min and

the supernatant material was removed and frozen until it

was analysed. Urine was collected after the first morning

void until time zero, and collections continued in timed 2 h

samples for 8 h from the start of consuming the drink. The

urine was kept cool and dark during collection and then

frozen until subsequent analysis. The volunteers were per-

mitted filtered tap water ad libitum. They were given two

plain biscuits after 1.25 h and again after 6.25 h after time

zero. A light lunch comprising two slices of wholemeal

bread, thinly spread with sunflower margarine, and 130 g

canned peaches in syrup was consumed after 3.25 h. This

food provided an additional 35 mg Ca, 48 mg Mg, 156 mg

phosphorus and 380 mg sodium.

The volunteers were seated in the laboratory for most of

the day, where they took part in light activities such as

reading, writing, knitting, sewing, card games and conversa-

tion. Most volunteers also had a short, leisurely walk out-

doors, which lasted for approximately 10 min. This was

typically completed at least 10 min before the first blood

sample after lunch.Serum PTH was measured by immunradiometric assay

(IRMA) using the Gamma-BCT intact PTH kit (Immunodiag-

nostics Systems Ltd, Boldon, UK). Normal serum PTH values

for adults aged 18 60y are 0.8 5.2pmol=l. Serum C-telo-

peptides (S-CTX) were measured by electrochemilumines-

cence immunoassay using the Roche Elecsys 1010 system

and a commercially available kit for b-Crosslaps (1972308

Roche Diagnostics, GmbH, D68298 Mannheim, Germany).

Normal serum CTX values for premenopausal women are of

the order of 0.28 ng=ml. Free deoxypyridinoline (DPD) was

measured using an automated chemiluminescence system

using the Chiron Diagnostics, ACS 180 DPD assay (Chiron

diagnostics, East Walpole, MA, USA). Normal urine-free DPDvalues for premenopausal women are 3.0 7.4nM DPD=mM

UCRN. Serum and urinary Ca and Mg were measured by

spectrophotometry (Cobas Fara II autoanalyser, Roche Diag-

nostics, Basel, Switzerland) using commercially available

kits. Calcium was measured using the Boehringer Mannheim

14893216 kit, and Mg was measured using the Boehringer

Mannheim 1489330 kit (Boehringer-Mannheim, Mannheim,

Germany). Urinary creatinine was measured by the Jaffe

reaction on an Abbott Aeroset Analyser (Abbott Laboratories,

Abbott Park, IL, USA).

The integrated areas under the curve for the change from

mean baseline for each serum measurement was calculated

using Simpsons rule. The 8 h excretion rates of Ca and Mgwere derived from the urinary concentrations of these

minerals and urine volume. Urine volume was calculated

from measurements of urine mass, made to the nearest 0.1 g

using a top pan balance (Sartorius Basiclite, BL3100, Sartorius

AG, Goettingen, Germany). The specific gravity of the urine

was not measured, but was assumed to be 1.02. Comparisons

between the start time for each drink and comparisons

between the areas under the curve were evaluated using a

one-way ANOVA and comparisons of means were done using

Tukeys pairwise comparisons. Comparisons between drinks

and the influence of time were evaluated using a cross-over

repeated measures analysis of variance (general linear models

procedure) and post hoc comparisons of means were done

using the Tukey Kramer test. Measurements were consid-

ered to be different if P

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change in serum Ca between the high-Ca drinks, but the

integrated increase in serum Ca was higher after each of

these drinks than after apple drink (P

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(P0.10), but it tended to be higher after each of the high

Ca drinks compared with apple. The 8 h Mg excretion rates

were 2410mg for apple, 3114mg TCP, 318mg for

high-Ca milk and 3317 mg for high-Ca skim milk enriched

with Mg. There was a significant positive correlation

between the total 8h excretion rates of Ca and Mg, each

expressed as mg=8 h (r0.53, P

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negative correlation between the integrated area under the

curve for change in serum PTH and 8 h Mg excretion

(r 70.31, P

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increases in erythrocyte Mg concentration (Doyle et al,

1999). However, a daily supplement of an amount of Mg

that was similar to the one we used (125 mg=day), provided

as Mg citrate malate for 3 weeks has been shown to have no

impact on intracellular erythrocyte concentration (Basso

et al, 2000).

In conclusion, TCP and high Ca milk with or without

additional Mg, led to an increase in serum Ca and decreases

in serum PTH and S-CTX, which confirms previous research

into the importance of calcium on bone metabolism. The

enrichment of high Ca skim milk with Mg had no additional

impact on serum PTH or bone resorption than high Ca skim

milk alone. Overall there were no differences in the effect of

the high calcium three drinks that we tested on calcium or

bone metabolism. Finally, because it is absorbed more slowly,

high calcium skim milk may be a better way to deliver

calcium than elemental calcium.

Acknowledgements

We are grateful to Dr Mark Morris, Chris Hartell SRN andPam Winger SRN, Student Health Services, Massey University

for their medical and nursing help with this study. We thank

Barbara-Kuhn Sherlock, Milk and Health Research Centre,

and Rob Crawford, New Zealand Dairy Research Institute, for

statistical advice, and Sue Croft, New Zealand Dairy Research

Institute, for help in providing the high Ca formulations.

Our thanks also go to Phil Pearce, Institute of Food, Nutri-

tion and Human Health, Massey University, Palmerston

North, James Yeo, Canterbury Health Laboratories, Christch-

urch and Sue Grant, Southern Community Laboratories,

Christchurch for doing the biochemical assays. The protocol

was peer-reviewed by Professor Ian Reid, Department of

Medicine, University of Auckland.

ReferencesAbraham GE & Grewal H (1990): A total dietary program emphasiz-

ing magnesium instead of calcium. Effect on the mineral densityof calcaneous bone in postmenopausal women on hormonaltherapy. J. Reprod. Med. 35, 503 507.

Angus RM, Sambrook PN, Pocock NA & Eisman JA (1988): Dietaryintake and bone mineral density. Bone Mineral 4, 265 277.

Basso LE, Ubbink JB, Delport R, Spies J & Hayward Vermaak WJ.(2000): Effect of magnesium supplementation on the fractionalintestinal absorption of 45 CaCl2 in women with a low erythrocytemagnesium concentration. Metabolism 49, 1092 1096.

Bjarnason NH, Henriksen EEG, Alexandersen P, Christgau S, Henrik-

son DB & Christiansen C (2002): Mechanism of circadian variationin bone resorption. Bone 30, 307 313.

Creedon A, Flynn A & Cashman K (1999): The effect of moderatelyand severely restricted dietary magnesium intakes on bonecomposition and bone metabolism in the rat. Br. J. Nutr. 82,63 71.

Dai LJ, Ritchie G, Kerstan D, Kang HS, Cole DE & Quamme GA(2001): Magnesium transport in the renal distal convolutedtubule. Physiol. Rev. 81, 51 84.

Dimai H-P, Porta S, Wirnsbirger G, Lindschinger M, Pamperl I,Dobnig H, Wilders-Truschnig M & Lau K-HW (1998): Daily oral

magnesium supplementation suppresses bone turnover in youngadult males. J. Endocrinol. Metab. 83, 2742 2748.

Doyle L, Flynn A & Cashman K (1999): The effect of magnesiumsupplementation on biochemical markers of bone metabolism orblood pressure in healthy young adult females. Eur. J. Clin. Nutr.53, 255 261.

Driessens FCM, Steidl L & Ditmar R (1990): Therapeutic effect ofmagnesium lactate supplementation on different forms of osteo-

porosis. Magnesium Bull. 12, 155

157.Freudenheim JL, Johnson NE & Smith EL (1986): Relationshipsbetween usual nutrient intake and bone-mineral content ofwomen 35 65 y of age: longitudinal and cross-sectional analysis.Am. J. Clin. Nutr. 44, 863 876.

Goddard M, Young G & Marcus R. (1986): Short-term effects ofcalcium carbonate, lactate, and gluconate on the calcium-para-thyroid axis in normal elderly men and women. Am. J. Clin. Nutr.44, 653 658.

Green JH, Richards J, Madigan J & Bunning R (2000): Bone resorptionresponses to magnesium-enriched high-calcium milk in postme-nopausal women. Bone 27(4 Suppl), 44S.

Horowitz M, Wishart JM, Goh D, Morris HA, Need AG & Nordin BE(1994): Oral calcium suppresses biochemical markers of boneresorption in normal men. Am. J. Clin. Nutr. 60, 965 968.

Hsin-Shan J Ju, Leung S, Brown B, Stringer MA, Leigh S, Scherrer C,Shepard K, Jenkins D, Knudsen J & Cannon R (1997): Comparison

of analytical performance and biological variability of three boneresorption assays. Clin. Chem. 43, 1570 1576.

Lau EMC, Woo J, Lam V & Hong A (2001): Milk supplementationof the diet of postmenopausal Chinese women on a lowcalcium intake retards bone loss. J. Bone Mineral Res. 16, 1704 1709.

Looker AC, Bauer DC, Chesnut CH, Gundberg CM, Hochberg MC,Klee G, Kleerekoper M, Watts NB & Bell NH (2000): Clinical use ofbiochemical markers of bone remodeling: current status andfuture directions. Osteoprosis Int. 11, 467 480.

McKane WR, Khosla S, Egan KS, Robins SP, Burritt MF & Riggs BL(1996): Role of calcium intake in modulating age-related increasesin parathyroid function and bone resorption. J. Clin. Endocrinol.Metab. 81, 1697 1698.

Miura T, Matsuzaki H, Suzuki K & Goto S (1999): Long term highintake of calcium reduces magnesium utilization in rats. Nutr. Res.19, 1363 1369.

New SA, Boton-Smith C, Grubb DA & Reid DM (1997): Nutritionalinfluences on bone mineral density: a cross sectional study inpremenopausal women. Am. J. Clin. Nutr. 65, 1831 1839.

Reginster JY, Denis D, Bartsch V, Deroisy R, Zegels B & Franchimont P(1993): Acute biochemical variations induced by four differentcalcium salts in healthy male volunteers. Osteoporosis Int. 3, 271 275.

Reid, IR. Hannan SF. Schooler BA & Ibbertson HK. (1986): The acutebiochemical effects of four proprietary calcium preparations. Aust.NZ J. Med. 16, 193 197.

Rubinacci A, Divieti P, Polo RM, Zampino M, Resmini G & Tenni R(1996): Effects of an oral calcium load on urinary markers ofcollagen breakdown. J. Endocrinol. Invest. 19, 719 726.

Rude RJ, Oldham SB & Singer FR (1976): Functional hyperpara-thyroidism and parathyroid hormone end-organ resistance inhuman magnesium deficiency. Clin. Endocrinol. (Oxford) 5, 209 224.

Sojka J, Wastney M, Abrams S, Lewis SF, Martin B, Weaver C &Peacock M (1997): Magnesium kinetics in adolescent girls deter-mined using stable isotopes: effects of high and low calciumintake. Am. J. Physiol. 273, R710 R715.

Stendig-Lindberg G, Tepper R & Leichter I (1993): Trabecular bonedensity in a two year controlled trial of peroral magnesium inosteoporosis. Magnesium Res. 6, 155 163.

Tranquilli AL, Lucino E, Garzetti GG & Romanini C (1994): Calcium,phosphorus and magnesium intakes correlate with bone mineralcontent in postmenopausal women. Gynecol. Endocrinol. 8,55 58.

PTH and bone resorption effects of milkJH Green et al

67

European Journal of Clinical Nutrition

7/29/2019 1601501 A

8/8

Tucker KL, Hannan MT, Chen H, Cupples LA, Wilson PFW & Kiel DP(1999): Potassium, magnesium and fruit and vegetable intakes areassociated with greater bone mineral density in elderly men andwomen. Am. J. Clin. Nutr. 69, 727 736.

Woo J, Swanminathan R, Lau E, MacDonald D, Pang CP & NordinBEC (1991): Biochemical effects of a single oral dose of calcium onbone metabolism in elderly Chinese women. Calcif. Tissue Int. 48,157 160.

Yang R-S, Liu T-K & Tsai K-S (1994): The acute metabolic effects oforal tricalcium phosphate and calcium carbonate. Calcif Tissue Int.55, 335 341.

PTH and bone resorption effects of milkJH Green et al

68

European Journal of Clinical Nutrition