Postprandial parathyroid hormone response to four calcium-rich foodstuffs1 2

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1 Postprandial parathyroid hormone response to four calcium-rich foodstuffs1 2 Merja UM K#{228}rkk#{228}inen, fan W Wiersma, and Christel fe Lamberg-Allardt ABSTRACT We studied the effects of four calcium-rich foodstuffs on postprandial parathyroid hormone secretion. Four hundred milligrams calcium from either Emmental cheese, milk, sesame seeds, spinach, or calcium salt (calcium lactate gluconate + calcium carbonate) or no additional calcium (control session) were given to nine female volunteers immediately after a first blood sample (at 9) in random order with a light standardized meal containing 37 mg Ca. Blood samples were taken at 9 (before the calcium load), 1, 11, 13, and 15 at every study session. Urine was collected during the sessions. Serum ionized calcium, phosphate, magnesium, intact parathyroid hormone, and urinary calcium excretion were measured. The serum ionized calcium concentration increased significantly after ingesting cheese (P =.4, contrast analysis) or calcium salt (P =.5, contrast analysis) compared with the control session. Compared with the control session, the serum phosphate concentration increased after the cheese session (P =.4, contrast analysis) and after the milk session (P =.2, contrast analysis). Calcium salt (P =.7, contrast analysis) and cheese (P =.2, contrast analysis) caused a significant decline in serum intact parathyroid hormone compared with the control session. The urinary calcium excretion with cheese was 141% (P =.1), with milk was 17% (P =.4), and with calcium salt was 75% (P =.2) above that of the control session. Our results show that calcium from sesame seeds and spinach does not cause an acute response in calcium metabolism. Our results indicate that fermented cheese could be a better dietary source of calcium than milk when the metabolic effects of the foodstuffs are considered. Am J Clin Nutr 1997;65: KEY WORDS Parathyroid hormone, calcium salt, fermented cheese, spinach, sesame seed, bioavailability, milk, magnesium, INTRODUCTION phosphate The importance for bone health of an adequate calcium intake during the life span has been emphasized. Whereas calcium is a building material needed in achieving maximal peak bone mass, supplemental calcium, when given to elderly people for example, may be considered a weak antiresorptive agent. Few studies show the effects of calcium from foodstuffs on bone and calcium metabolism and bone density. It has been shown that 2-y milk supplementation improved calcium balance in postmenopausal women (1) and that 3-y milk supplementation prevented a decrease in bone mineral density in premenopausal women (2). Some retrospective studies have shown a positive effect of milk intake in youth on bone mineral density later in life (3). Parathyroid hormone (PTH) is one of the major regulators of calcium and bone metabolism. PTH increases bone resorption and acutely decreases some stages of bone formation (4). An acute oral calcium load increases the serum ionized calcium concentration and suppresses PTH secretion (5-7) in a dosedependent manner (8, 9). Calcium from foodstuffs may have acute effects on calcium and bone metabolism, but this has not been studied. These metabolic effects could be important when different dietary calcium sources are discussed and recommended. The absorbability of calcium is one factor affecting calcium utilization (1). Oxalate and phytate may decrease calcium absorption ( 1 1, 12), whereas lactose may enhance it ( 13). In addition, phosphate ( 14, 15) and magnesium (16) have direct effects on calcium and bone metabolism. This study investigated the acute metabolic effects of some calcium-rich foodstuffs on calcium metabolism. SUBJECTS AND METHODS Subjects and protocol Nine healthy female volunteers using oral contraceptives, aged y, took part in the trial. All subjects gave their informed consent to the procedures, which were in accord with the Helsinki Declaration. The subjects took no medications known to affect calcium and bone metabolism. The basic characteristics of the subjects are presented in Table 1. The amount of calcium administered in this study was chosen on the basis of a pilot study in which we noted a significant response in PTH secretion to 25 mg Ca administered as calcium salt (681 mg calcium lactate gluconate, 3 mg calcium carbonate, 135 mg acid citrate anhydrous, and 4 mg From the Calcium Research Unit, Department of Applied Chemistry and Microbiology, University of Helsinki; the Minerva Foundation Institute for Medical Research, Helsinki; and the Department of Nutrition, University of Wageningen, Netherlands. 2 Address reprint requests to C Lamberg-Allardt, Calcium Research Unit, Department of Applied Chemistry and Microbiology, P Box 27, FIN-14 University of Helsinki, Finland. Christel.Lambergallardt@helsinki.fi. Received June 25, Accepted for publication January 1, Downloaded from ajcn.nutrition.org at PENNSYLVANIA STATE UNIV PATERNO LIBRARY on May 9, Am J Clin Nutr l997;65: Printed in USA American Society for Clinical Nutrition

2 PTH RESPONSE TO FOUR CALCIUM-RICH FOODSTUFFS 1727 TABLE 1 Basic characteristics of the study group Age(y) 26±1 Height (m) 1.65 ±.2 Weight (kg) 55 ± 3 Energy intake (MI) 8.7 ±.6 (kcal) 286 ± 146 Calcium intake (mg) 1293 ± 13 Phosphate intake (mg) 1554 ± 135 I ± SEM; n = 9 women. aspartame, corresponding to 1 g Ca2 ; Laboratoires Sandoz, SARL, France). Because the calcium in foodstuffs may not be absorbed as easily as calcium salts, the calcium dose we chose was 4 mg/portion. We wanted to keep the portion sizes as small as possible. Four foodstuffs were selected for the experiment: fermented cheese (Emmental type, 3% fat), milk ( 1.5% fat), whole sesame seeds, and spinach. Milk and cheese are important sources of dietary calcium. Sesame seeds are considered to be a rich source of calcium in a vegetarian diet. Only whole sesame seeds, however, contain a large amount of calcium; decorticated sesame seeds contain only 1% of the calcium in whole seeds. The calcium content of spinach is high, but the absorbability of this calcium is poor (17). The biochemical responses to spinach calcium may be small. Calcium salt was used as a standard for calcium bioavailability. In addition, the daily variation in PTH secretion was assessed by a control session. The order of ingestion of foodstuffs, calcium salt, and the control was assigned randomly. The calcium and phosphate contents of the foodstuffs were analyzed in frozen specimens by routine methods before the trial (Valio Ltd. R&D Laboratory Services, Chemical Division, Helsinki). The portion sizes were 333 g milk, 43 g Emmental cheese, 36 g sesame seeds, and 19 g spinach. Each portion contained 4 mg Ca. The phosphate contents of the portions were 254 mg for cheese, 286 mg for milk, 23 1 mg for sesame seeds, and 86 mg for spinach. The calculated protein contents were 1 1 g for milk and 12 g for cheese. The foodstuffs were stored at -2 #{176}C until divided into portions. Sampling The subjects fasted overnight. The first blood sample was drawn at 9 at every study session. After the first blood sample, a light meal (.2 L orange juice, 5 g butter,.2 L tea, 4 g sugar, 5 g cucumber, 25 g white bread, and.2 L water, except with milk) together with the experimental foodstuff or calcium salt was served at each session and at the control session. The next blood samples were drawn at 1, 11, 13, and 15. A standardized lunch, which consisted of.2 L orange juice, 5 g margarine, 5 g white bread, 5 g carrot, and 275 g potato and meat casserole, was served immediately after the blood sample at 13. The breakfast contained 37 mg Ca, 71 mg P, 5 g fat, and 2 g protein, and the lunch contained 4 mg Ca, 29 mg P. 12 g fat, and 12 g protein by calculation. Serum was separated from the blood samples. Urine was collected 24 h before the test and during the study every 2 h from 7 to 15. The serum and urine samples were refrigerated at -2 #{176}C until analyzed. Biochemical analyses The serum ionized calcium concentration was measured within 9 mm after blood sampling with a selective ion analyzer (ISE Ca k/ph Analyzer 634; Ciba Coming Diagnostics, Halstead, United Kingdom). The ionized calcium concentration was adjusted to ph 7.4. Serum total calcium, phosphate, magnesium, and urinary calcium, phosphate, and magnesium concentrations were measured by routine laboratory methods. The concentration of intact PTH (ipth) was analyzed with the Allegro intact PTH kit from Nichols Institute (San Juan Capistrano, CA). The intraassay CV for ipth was 1 % and the interassay CV was 3.7%. Statistical analyses The data are expressed as means ± SEMs. After testing for normality, logarithmic transformations were used when necessary. Analysis of variance (ANOVA) with repeated measures was used to test differences in biochemical responses among the six study sessions. If the sphericity assumption was not met, Hyunh-Feldt adjusted P values were used. If a significant difference was found with repeated-measures ANOVA, contrast analysis was used to compare the response curves of study sessions with the control session. Analyses were made with BMDP statistical software for VAX/VMS minicomputer (18). The results are presented as two panels in the figures only for readability. RESULTS The analysis of the response curves showed a significant difference in ionized calcium concentration (Figure 1) among the study sessions (P =.3). The concentration of ionized calcium increased significantly after ingesting cheese (P =.4) and calcium salt (P =.5) and tended to increase after milk (P =. 1 ), whereas no difference was found between the spinach and sesame seed sessions compared with the control session. No differences were found in the serum total calcium concentration among the study sessions. The response curves of the serum phosphate concentrations were affected (P =.1) by the foodstuffs. Compared with the control session, a significant increase was found in serum phosphate concentration (Figure 2) after the cheese and milk sessions (P =.4, P =.2, respectively). Sesame seeds and spinach did not affect the serum phosphate concentration. No differences in serum magnesium concentrations were found among the study sessions. The ipth secretion (Figure 3) was affected by the test substances (P =.5). The decrement in PTH secretion after ingesting calcium salt (P =.7) and cheese (P =.2) were significant compared with the control session. The other foodstuffs did not significantly affect PTH secretion. Urinary calcium excretion, which in the short term reflects calcium absorption (Figure 4), was significantly affected by some of the tested foodstuffs (P.5). The urinary excretion ofcalcium with cheese was 141% (P =.1), with milk was 17% (P =.4), and with calcium salt was 75% (P =.2) above that of the control session. No significant changes were found in urinary calcium excretion after the ingestion of sesame seeds or spinach. Although there was a Downloaded from ajcn.nutrition.org at PENNSYLVANIA STATE UNIV PATERNO LIBRARY on May 9, 216

3 1728 KARKKAINEN ET AL -J + 1.E U).2 C- _C E. Cl) 12 OC -CO E. Cl) 1. A I I I I -J C Cl).2 - C- C.C FIGURE 1. Changes in serum ionized calcium concentration (i ± SEM: n = 9 women) during the study sessions. The dose of calcium ingested from cheese, milk, spinach, and calcium salt (calcium lactate gluconate + calcium carbonate) was 4 mg and it was consumed with a light standardized breakfast containing 37 mg Ca. During the control session, no additional calcium was consumed. For readability, the results of the milk ( #{149} ) and cheese (Y) sessions are presented in panel A. The spinach (U) and sesame seed (A) sessions are presented in panel B. The control (L) and calcium salt () sessions are the same in both panels. Significant differences among study sessions, P =.3 (repeated-measures ANOVA); asignificantly different from control session, P =.5 (contrast analysis): bp.4 (contrast analysis). trend for the urinary excretion of phosphate to increase after cheese and milk, the changes were not significant. Urinary magnesium excretion was affected by the tested foodstuffs (P =.2), being above that of the control session with calcium salt (P =.4) and cheese (P =.4). DISCUSSION To our knowledge, this is the first study in which the acute metabolic effects of calcium-rich foodstuffs on calcium metabolism have been investigated. The response in ionized calcium concentration differed among the foodstuffs tested. Calcium salt increased the serum ionized calcium concentration, as was shown earlier (9, 19). There was a remarkable difference among the foods studied. The ingestion of fermented cheese increased the serum ionized calcium concentration, but the effect of milk on serum ionized calcium concentration was not significant. One factor affecting the response in ionized cal- 7 -I E Cl) OC B FIGURE 2. Changes in serum phosphate concentration concentration ( ± SEM; n = 9 women) during the study sessions. The dose of calcium ingested from cheese, milk, spinach, and calcium salt (calcium lactate gluconate + calcium carbonate) was 4 mg and it was consumed with a light standardized breakfast containing 37 mg Ca. During the control session, no additional calcium was consumed. For readability, the results of the milk ( #{149} ) and cheese (V) sessions are presented in panel A. The spinach (#{149}) and sesame seed (A) sessions are presented in panel B. The control (U) and calcium salt () sessions are the same in both panels. Significant differences among the study sessions, P =.1 (repeated-measures ANOVA); asignificantly different from control session, P =.4 (contrast analysis); bp.2 (contrast analysis). cium concentration after a calcium load is the amount of calcium absorbed (8). Milk calcium has been shown to be absorbed easily in human experiments using extrinsically radiolabeled milk (2). To our knowledge, the bioavailability of calcium from fermented cheese has not been investigated, but the bioavailability of calcium from fresh cheese (rennet-precipitated casein) has been reported to be equal to that of milk (2). Some studies have reported that lactose enhances calcium absorption (13), but this finding has not been supported by others (21). Although we did not measure calcium absorption, our results do not support the finding that lactose enhances calcium absorption because the increase in ionized calcium after the ingestion of cheese, which contains minimal amounts of lactose, was more pronounced than after milk. It may be that the long bacterial fermentation of cheese affects calcium absorbability. Another possibility is that the greater fat content of cheese slowed gastric emptying and thus enabled more calcium to be absorbed. 7 Downloaded from ajcn.nutrition.org at PENNSYLVANIA STATE UNIV PATERNO LIBRARY on May 9, 216

4 PTH RESPONSE TO FOUR CALCIUM-RICH FOODSTUFFS 1729 I I C Cl)W Cl).;. Co C C CO)L. oe a. E E I 2C - Cl) C.;. ce CO. 3 4 FIGURE 3. Changes in serum intact parathyroid hormone (ipth) concentration ( ± SEM, n = 9 women) during the study sessions. The dose of calcium ingested from cheese, milk, spinach, and calcium salt (calcium lactate gluconate + calcium carbonate) was 4 mg and it was consumed with a light standardized breakfast containing 37 mg Ca. During the control session, no additional calcium was consumed. For readability, the results of the milk ( #{149} ) and cheese (V) sessions are presented in panel A. The spinach (U) and sesame seed (A) sessions are presented in panel B. The control (U:]) and calcium salt () sessions are the same in both panels. Significant differences among the study sessions, P.5 (repeatedmeasures ANOVA); usignificantly different from control session, P.7 (contrast analysis); b p.2 (contrast analysis). It has been shown that serum ionized calcium declines rapidly after a phosphate load (15, 22). The phosphate content was almost equal in the portions of milk, cheese, and sesame seeds. The amount of phosphate in the foods was small and it seems unlikely that it could have affected the serum ionized calcium concentrations to a large extent, although serum phosphate rose significantly after ingestion of cheese and milk. Spinach and sesame seeds did not affect the serum ionized calcium concentration. The bioavailability of calcium from spinach is poor as shown in a calcium absorbability experiment (17). The bioavailability of calcium from sesame seeds is also poor, as indicated by a rat experiment with the tibia calcium content used as an indicator of bioavailability (23). These facts explain the low response in serum ionized calcium concentration after ingestion of these two foodstuffs. The serum P FH concentration decreases when serum ionized calcium concentration increases, which in turn is affected in a dose-dependent manner by the amount of calcium that is absorbed from the intestine (8, 9). Of the foodstuffs studied, only FIGURE 4. Postload urinary calcium excretion after subjects consumed a light standardized breakfast containing 37 mg Ca with no additional calcium (U:l) or 4 mg Ca from either calcium lactate gluconate + calcium carbonate (U), cheese (U). milk (a). sesame seeds (a), or spinach (a). asignificantly different from control session, P.2; bp.1: CP.4. fermented cheese decreased the PTH concentration significantly, which is in line with the response in the serum ionized calcium concentration. The average decrease in serum PTH after the milk session was 2% compared with the control session, which may have been significant with a larger number of subjects. This response is small and was reached after a 4-mg dose of calcium, which, in an everyday situation, would be considered large when milk is the source. A normal dose would be a glass of milk (.2 L) corresponding to =24 mg Ca, which would give an even smaller PTH response. Some other nutrients and factors could directly affect PTH secretion. Phosphate increases PTH secretion (14, 15) and magnesium decreases (16) it. This finding could not explain the different response of PTH to milk and cheese, however, because the amount of phosphate was similar in these foodstuffs, and the calculated amount of magnesium in the portions was < 4 mg and did not affect the serum magnesium concentration. In a controlled situation there is a strong correlation between the acute intake of calcium and urinary calcium excretion (24). In our study, urinary calcium excretion did not increase after ingestion of sesame seeds and spinach, which confirms the low absorption of calcium from these products. Urinary calcium excretion after the ingestion of milk and cheese was equal to that after calcium salt, which indicates that calcium was absorbed equally from these. Why was calcium excretion affected by both milk and cheese, whereas the ionized calcium and PTH responses were not affected by milk? The reason for this is unknown. Serum phosphate changed in the same manner after the ingestion of both foodstuffs. The meals themselves could not have influenced the biochemical response because the same meals were served at all study sessions and at the control session. Can recommendations be given for foodstuffs that are especially good for bone health on the basis of the results in this experiment? The results of our study indicate that the metabolic effect is different among different foodstuffs. Calcium bioavailability in spinach and sesame seeds seems to be poor. However, grinding could increase the bioavailability of calcium in sesame seeds, but this must be confirmed by more studies. Although calcium in milk has been shown to be absorbed as well as calcium carbonate in absorbability studies Downloaded from ajcn.nutrition.org at PENNSYLVANIA STATE UNIV PATERNO LIBRARY on May 9, 216

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