Genetic influences on bone density: Physiological correlates of vitamin D receptor gene alleles in premonopausal women

0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society

Vol. 83, No. 3 Printed in U.S.A.

LETTERS TO THE EDITOR Another Potential Use of Troglitazone in NoninsulinDependent Diabetes Mellitusa To the editor:

Jun Minamikawa, MD, Mika Yamauchi, MD, Daisuke Inoue, MD, and Hiroyuki Koshiyama, MD Hyogo Prefectural Amagasaki Hospital Hyogo 660, Japan

We read with interest the article by Izumino et al. (1), which suggests a therapeutic use of troglitazone, an insulin sensitizer, in Werner’s syndrome as well as in noninsulin-dependent diabetes mellitus (NIDDM). We here report another possible benefit of troglitazone treatment: prevention of atherosclerosis. We measured the intimal and medial complex thickness (IMT) of common carotid artery using B-mode ultrasound technique to evaluate early atherosclerotic lesions (2). First we investigated the relationship between IMT and urinary C-peptide levels (uCPR) or insulin dosage in 106 Japanese subjects with NIDDM [52 males and 54 females, age 62.5 yr (se 0.9) yr]. Eighty-one of them were receiving insulin treatment, and the others were taking sulfonylureas. u-CPR of 24-hr urine samples were measured with a radioimmunoassay kit and were expressed as a mean value in three consecutive days. IMT values showed a positive correlation with both u-CPR (r 5 0.655, P , 0.0001) and insulin dosage, calculated as an average dose per day (r 5 0.399, P , 0.005). The correlation remained significant after adjusting for HbAIc, body mass index, age, serum total cholesterol, and triglyceride levels. Second, we examined the effect of short-term treatment with troglitazone (400 mg daily for 3 months) on IMT in 33 patients with NIDDM. Before troglitazone treatment they had been treated with sulfonylureas (32 glibenclamide and 1 gliclazide), which were continued in the same doses during the troglitazone treatment. Thirty-two diabetic subjects (29 receiving sulfonylureas and 3 diet alone) were examined as control group. The group given troglitazone showed a significant decrease in IMT after 3 months [IMT change: 20.196 mm (se 0.082) vs. control 0.034 mm (se 0.010), P , 0.01]. There was no relation between a decrease in IMT and a decline in HbAIc. Our finding indicated an association of IMT with both endogenous and exogenous insulin in NIDDM. It is in contrast with a previous study, which failed to demonstrate an association of serum C-peptide with IMT in NIDDM (3), but is compatible with another report indicating an association of fasting insulin and IMT in normal subjects (4). It is also intriguing that we found an analogous association between lumbar bone mineral density and endogenous or exogenous insulin in NIDDM (5). It is possible that the association of IMT with insulin may reflect a direct or indirect atherogenic action of insulin on the vascular wall. However, troglitazone markedly decreased IMT, suggesting that the correlation of IMT and insulin represents a relationship between atherosclerosis and insulin resistance rather than the actions of insulin. Negative association has recently been reported between IMT and insulin sensitivity (6). Alternatively, it is possible that insulin has both atherogenic and antiatherogenic actions, but insulin resistance may selectively inhibit the antiatherogenic action (7), which can be reversed by troglitazone treatment. However, actions of troglitazone other than those as an insulinsensitizer, such as antioxidant activity (8), cannot be totally excluded. Whatever the mechanisms, the present preliminary result suggests a potent inhibitory action of troglitazone on atherosclerosis, which is compatible with a study in the rat (9). Since Werner’s syndrome is characterized with progeria (1), it is of interest to investigate whether IMT is increased and whether troglitazone may decrease IMT in this disorder. It is also to be elucidated whether troglitazone may prevent restenosis after coronary angioplasty in NIDDM. The authors are grateful to Dr. Hougaku for his technical advice.

References

a Received October 27, 1997. Address correspondence to: Dr. Hiroyuki Koshiyama, Division of Endocrinology and Metabolism, Department of Internal Medicine, Hyogo Prefectural Amagasaki Hospital, 1-1-1 Higashi-Daimotsu-cho, Amagasaki, Hyogo 660, Japan.

1. Izumino K, Sakamaki H, Ishibashi M, et al. 1997 Troglitazone ameliorates insulin resistance in patients with Werner’s syndrome. J Clin Endocrinol Metab. 82:2391–2395. 2. Fukunaga Y, Minamikawa J, Inoue D, Koshiyama H., Fujisawa I. 1997 Pseudoacromegaly and hyperinsulinemia: a possibility of premature atherosclerosis? (Letter). J Clin Endocrinol Metab. 82:3515–3516. 3. Pujia A, Gnasso A, Irace C, Colonna A, Mattioli PL. 1994 Common carotid arterial wall thickness in NIDDM subjects. Diabetes Care. 17:1330 –1336. 4. Folsom AR, Eckfeldt JH, Weitzman S, et al. 1994 Relation of carotid artery wall thickness to diabetes mellitus, fasting glucose and insulin, body size, and physical activity. Stroke. 25:66 –73. 5. Fukunaga Y, Minamikawa J, Inoue D, Koshiyama H. 1997 Does insulin use increase bone mineral density in patients with non-insulin-dependent diabetes mellitus? Arch Intern Med. 157:2668 –2669. 6. Howard G, O’Leary DH, Zaccaro D, et al. 1996 Insulin sensitivity and atherosclerosis. Circulation. 93:1809 –1817. 7. Feener EP, King GL. 1997 Vascular dysfunction in diabetes mellitus. Lancet. 350:SI9 –13. 8. Cominacini L, Garbin U, Pastorino AM, et al. 1997 Effects of troglitazone on in vitro of LDL and HDL induced by copper irons and endothelial cells. Diabetologia. 40:165–172. 9. Law RE, Meehan WP, Xi X-P, et al. 1997 Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 98:1897–1905.

Troglitazone Ameliorates Insulin Resistance in Patients with Werner’s Syndrome—Author’s Responseb To the editor: Thank you very much for the opportunity to respond to Dr. Minamikawa’s letter (above). The data is interesting, providing the intimal and medial complex thickness (IMT) positively correlate with both endogenous and exogenous insulin in NIDDM patients. Furthermore, troglitazone treatment (400 mg per day for 3 months) significantly resulted in decrease in IMT (20.196 6 0.082 mm). The results associated with the first part have been demonstrated in our study showing that the NIDDM patients suffering from arteriosclerosis obliterans (ASO) were observed to be more insulin resistant than those not suffering from ASO (1). In that study, insulin resistance was assessed by the short insulin tolerance test’s K index (Kitt) (2.16 6 0.16 vs. 3.00 6 0.13%/min, with ASO vs. without, respectively). Although Dr. Minamikawa did not mention in his letter how much insulin resistance the patients had, it is quite possible to speculate that higher levels of plasma insulin may be the reflection by the insulin resistance in their patients. For the second part, we do not have sufficient data indicating the troglitazone’s preventive or inhibitory effects on atherosclerosis progression in the patients with Werner’s syndrome in our study. We fully agree with the idea that trials of troglitazone in the treatment of Werner’s syndrome should be conducted to test for the attenuation of atherosclerosis. Skin ulcers as clinical manifestation were found in three out of five patients with Werner’s syndrome in our study and not in the remainder (2). Therefore the sensitive examination, for example IMT, should be required to find early changes of atherosclerosis in these patients, and they deserve to have treatment with troglitazone to evaluate the antiatherosclerotic effect of this drug. The research on the pathogenesis of atherosclerosis found in Werner’s syndrome is just beginning to reveal answers. Hypercoagulable condition b Received December 4, 1997. Address correspondence to: Hironori Yamasaki, M.D., The First Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto Nagasaki, 852 Japan.

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was observed as manifested by an increase in the level of tissue plasminogen activator (t-PA) and PA inhibitor-1 (PAI-1), and by decrease in the level of thrombomodulin (TM) in 9 patients with Werner’s syndrome (3). Thus, complex risk factors including hyperinsulinemia, insulin resistance, and hypercoagulation would be expected to contribute to the progression of atherosclerosis in Werner’s syndrome as well as in NIDDM. Although exact biochemical action of troglitazone has not been determined, the application of this drug to Werner’s syndrome would delineate possible mechanisms by which anti-atherosclerotic action would be evoked with this. Hironori Yamasaki, Kiyohiro Izumino, Hirofumi Takino, Shigenobu Nagataki, and Yoshihiko Yamaguchi Nagasaki University School of Medicine Nagasaki, Japan

References 1. Matsumo K, Miyake S, Yano Y, et al. 1997 Insulin resistance and arteriosclerosis obliterance in patients with NIDDM. Diabetes Care. 20:1738 –1743. 2. Izumino K, Sakamaki H, Ishibashi M, et al. 1997 Troglitazone ameliorates insulin resistance in patients with Werner’s syndrome. J Clin Endocrinol Metab. 82:2391–2395. 3. Goto M, Kato Y. 1995 Hypercoagulable state indicates an additional risk factor for atherosclerosis in Werner’s syndrome. Thromb Haemostasis. 73:576 –578.

Longitudinally Sampled Human Plasma Leptin and Cortisol Concentrations Are Inversely Correlatedc To the editor: Haffner et al. (1) reported that leptin concentrations were not significantly related to cortisol. In that study the authors measured fasting levels of cortisol and leptin at one time point in 87 normoglycemic men. It has long been known that plasma cortisol concentrations exhibit clinically relevant ultradian and circadian fluctuations that can be altered in disease states such as major depression (2). More recently, our group (3) and others (4, 5) have shown that plasma concentrations of leptin have pulsatility and diurnal variation, which seem to be of biological relevance: Matkovic et al. (6) have shown that a blunted nocturnal rise in leptin levels correlates with weight gain. Because both cortisol and leptin exhibit statistically significant ultradian and diurnal fluctuation that are clinically relevant, it is inadequate to rely on single fasting measurements to assess a relationship between these hormones. In our own studies we have shown a highly significant inverse relationship between the variability of simultaneous 1,242 measurements of cortisol and leptin in 6 normoglycemic men who were sampled every 7 min for 24 h (Pearson correlation: r 5 0.764; P , 1029) (3). These data are consistent with the findings of Ahima et al. (7), that in rodents leptin administration blunts fasting-induced increases in cortisol levels, and with the findings of Bornstein et al. (8), that leptin acts directly in the adrenal gland to suppress cortisol production. The effects of leptin on hypothalamic-pituitary-adrenal function indicate a mechanism by which a pulsatile peripheral signal of nutritional status may regulate stress-related endocrine function and behavior. Because the levels of leptin, adrenocorticotropic hormone, and cortisol are highly pulsatile, frequently-sampled longitudinal measurements are required for the study of relations between leptin and cortisol. Therefore, Haffner et al.’s conclusion that leptin levels in healthy men are not significantly related to cortisol is most likely erroneous, and reflects insufficient sampling. Julio Licinio Clinical Neuroendocrinology Branch NIMH, NIH Bethesda, MD 20892

References 1. Haffner SM, Miettinen H, Karhapaa P, Mykkanen L, Laakso M. 1997 Leptin concentrations, sex hormones, and cortisol in nondiabetic men. J Clin Endocrinol Metab. 82:1807–1809. 2. Sachar EJ, Hellman L, Fukushima DK, Gallagher TF. 1970 Cortisol producc

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LETTERS TO THE EDITOR

Received July 25, 1997. Address correspondence to: Julio Licinio, Clinical Neuroendocrinology Branch, NIMH, NIH, Bldg. 10/2D46, 10 Center Drive, MSC 1284, Bethesda, Maryland 20892-1284.

3. 4. 5. 6. 7. 8.

tion in depressive illness: a clinical and biochemical clarification. Arch Gen Psychiatry. 23:289 –298. Licinio J, Mantzoros C, Negra˜o AB, et al. 1997 Human leptin levels are pulsatile and inversely related to pituitary-adrenal function. Nat Med. 3:575–579. Sinha MK, Ohannesian JP, Heiman ML, et al. 1996 Nocturnal rise of leptin in lean, obese, and noninsulin-dependent diabetes mellitus subjects. J Clin Invest. 97:1344 –1347. Sinha MK, Sturis J, Ohannesian J, et al. 1996 Ultradian oscillations of leptin secretion in humans. Biochem Biophys Res Commun. 228:733–738. Matkovic V, Ilich JZ, Badenhop NE, et al. 1997 Gain in body fat is inversely related to the nocturnal rise in serum leptin level in young females. J Clin Endocrinol Metab. 82:1368 –1372. Ahima RS, Prabakaran D, Mantzoros C, et al. 1996 Role of leptin in the neuroendocrine response to fasting. Nature. 382:250 –252. Bornstein SR, Uhlmann K, Haidan A, Ehrhart BM, Scherbaum WA. 1997 Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes. 46:1235–1238.

Leptin Concentrations, Sex Hormones, and Cortisol in Nondiabetic Men—Authors’ Responsed To the editor: Thanks to Dr. J. Licinio for his letter on our report (1). We are, of course, well aware that leptin levels have a diurnal variation. We have previously published that leptin levels vary diurnally in diabetic subjects (2) and that glibenclamide (but not acarbose) raises leptin concentrations. In that report we cited the report of Sinha et al. (2) but were not aware of your report of Licinio et al. (3). We did, however, cite evidence (4, 5) for the circadian rhythm of cortisol in our report as one of the explanations for the circadian variation in leptin (2). So we are certainly not unaware of the circadian rhythm of leptin and indeed cortisol. We were surprised by the letter as our report treats almost exclusively the observation that sex hormones are unlikely to explain the sex difference in leptin levels. We acknowledge that repeated sampling may improve precision for both sex hormones and cortisol, but our correlations of both sex hormones and cortisol with obesity (i.e. BMI) are in the range previously reported for studies that used multiple sampling. So we stand by the precision of our laboratory measurements. We admire both the financial resources of the investigators and the fortitude of the 6 subjects to have 1242 measurements made. We acknowledge that leptin is involved in cortisol release, although we suspect that the association of leptin with cortisol is much weaker than its relation to adiposity; thus, carefully controlled pharmacologic studies or very frequent sampling (as were done by Licinio et al. (2)) are necessary to elucidate this association. S. M. Haffner and H. Miettinen University of Texas Health Sciences Center San Antonio, Texas 78284 P. Karhapa¨a¨, L. Mykka¨nen, and M. Laakso Kuopio University Hospital Kuopio, Finland

References 1. Haffner SM, Miettinen H, Karhapa¨a¨ P, Mykka¨nen L, Laakso M. 1997 Leptin concentrations, sex hormones, and cortisol in nondiabetic men. J Clin Endocrinol Metab. 82:1807–1809. 2. Haffner SM, Hanefeld M, Fischer S, Fu¨cker K, Leonhart W. 1997 Glibenclamide, but not acarbose, increases leptin concentrations parallel to changes in insulin in subjects with NIDDM. Diabetes Care. 20:1430 –1434. 3. Licinio J, Mantzoros C, Negra˜o AB, et al. 1997 Human leptin levels are pulsatile and inversely related to pituitary-adrenal function. Nat Med. 3:575–579. 4. Weitzman ED, Zimmerman JC, Czeisler CA, Ronda JM. 1983 Cortisol secretion is inhibited during sleep in normal man. J Clin Endocrinol Metab. 56:352–358. 5. Lejeune-Lenain C, Van Cauter E, Desir D, Beyloos M, Franckson JRM. 1987 Control of circadian and episodic variations of adrenal androgens secretion in man. J Endocrinol Invest. 10:267–276.

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Received November 20, 1997. Address correspondence to: Steven M. Haffner, Department of Internal Medicine, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7873.

LETTERS TO THE EDITOR Genetic Influences on Bone Density: Physiological Correlates of Vitamin D Receptor Gene Alleles in Premenopausal Women. Notification of Genotype Correctionse To the editor: It has come to our attention that there were some errors in the genotyping used in our study of the genetic influences on bone density (1). The subjects were drawn in part from a larger study (Nature: 1994: 367: 284 –287. Prediction of Bone Density by VDR Alleles) in which errors have been reported. We would like to notify the scientific community of these changes and of the following findings with the corrected genotyping. Recently we have noted some errors in vitamin D receptor genotyping, which affected the assignment of the individuals studied. Thus genotyping was repeated by two independent technicians in a blinded fashion, and additional healthy premenopausal subjects were recruited to increase the sample size to 12 in the BB genotype and 16 in the bb genotype. Our original findings of differences in osteocalcin and 1,25-dihydroxyvitamin D between genotypes were not confirmed in this reanalysis. However, as outlined below, iPTH levels were higher and basal urinary calcium excretion greater in the BB genotype, as was the rate and extent of PTH suppression with calcitriol treatment. This was contrary to the original report of a greater PTH response in the bb genotype. Also urinary excretion of calcium and hydroxyproline rose significantly in response to 1,25-dihydroxyvitamin D, but only in the BB homozygotes. At baseline there were no significant differences between the two genotypes with regard to height, weight, age, bone density, or in osteocalcin or 1,25-dihydroxyvitamin D levels. Subjects in the BB genotype had significantly higher intact parathyroid hormone (iPTH) levels (3.3 6 0.4 vs. 2.3 6 0.3 pmol/L, mean 6 sem, P 5 0.03), with a trend for lower urinary calcium excretion (0.22 6 0.07 vs. 0.39 6 0.06 mmol/mmol, P 5 0.08). Serum 1,25-dihydroxyvitamin D rose to a similar extent in both genotypes after calcitriol stimulation, and serum ionized calcium, phosphate and TmPO4 also responded similarly in both genotypes. The decrease in iPTH was greater in the BB genotype (21.3 6 0.2 vs. 20.45 6 0.2 pmol/L, BB vs. bb, P 5 0.04) and occurred earlier (day 2 vs. day 4). Serum osteocalcin levels increased in both genotypes, but the apparently greater rise by day 2 in bb vs. BB individuals did not achieve significance (3.6 6 0.6 mg/L vs. 1.8 6 0.9 mg/L, P 5 0.1). An increase in urinary calcium/creatinine (Ca/Crt) and hydroxyproline excretion in response to 1,25-dihydroxyvitamin D was apparent only in the BB genotype; differences were (BB, 0.08 6 0.04 mmol/mmol, P 5 0.0001, vs. bb, 0.02 6 0.04 mmol/mmol, P 5 0.07) and (BB, 2 6 2, P 5 0.04, vs. bb, 1 6 1 mmol/mmol, P 5 0.2), respectively. Thus after correction of genotyping errors and increasing the sample size, a VDR genotype related difference in both basal calcium homeostasis and response to calcitriol stimulation was still observed in healthy premenopausal women. Although basal differences in 1,25-dihydroxyvitamin D levels were not detected between VDR homozygotes in the present study, studies with larger sample sizes have reported higher 1,25-dihydroxyvitamin D and osteocalcin levels in the BB allele (2). Higher levels of iPTH in the BB genotype have also recently been reported in hyperparathyroid patients (3). Overall these reanalyzed data are consistent with a difference in response to calcitriol according to VDR genotype. Gabrielle Howard, Tuan Nguyen, Nigel Morrison, Takako Watanabe, Philip Sambrook, John Eisman, and Paul Kelly The Garvan Institute of Medical Research Sydney, New South Wales 2010, Australia

References 1. Howard G, Nguyen T, Morrison N, et al. 1995 Genetic influences on bone density: physiological correlates of vitamin D receptor gene alleles in premenopausal women. J Clin Endocrinol Metab. 80:2800 –2805. 2. Tokita A, Matsumoto H, Morrison N, et al. 1996 Vitamin D receptor alleles, e Received December 8, 1997. Address correspondence to: Dr. Gabrielle Howard, The Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.

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bone mineral density and turnover in premenopausal Japanese women. J Bone Miner Res. 11:1003–1009. 3. Carling T, Kindmark A, Hellman P, et al. 1995 Vitamin D receptor genotypes in primary hyperparathyroidism. Nature Med. 1:1309 –1311.

Hemodynamic Effects of Parathyroid HormoneRelated Peptide: Is There a Pathophysiological Relevance?f To the editor: We read with interest the paper by M. Wolzt et al. (1) related to the hemodynamic effects of parathyroid hormone-related peptide (PTHrP) in man. These authors observed a dose-dependent increase in pulse rate, renal plasma flow, and hand vein diameter without any change in blood pressure in response to the infusion of the PTHrP analog PTHrP (1–34). Although this interesting study is the first to evaluate the effect of PTHrP in man, some conclusions are overdrawn beyond the information provided, mainly because of the lack of hemodynamic and hormonal assessment. As heart rate increased but blood pressure remained unchanged, determination of cardiac output and systemic vascular resistances would have allowed to attribute a peripheral vasodilating effect definitely to PTHrP. Measurement of catecholamines would have helped to differentiate tachycardia due to reflex baroreceptor responses from a direct chronotropic effect of PTHrP, as suggested in experiments using isolated rat hearts (2). The authors also claim that no effect was noted on parameters of cardiac inotropic performance, which were not evaluated as such by the present study even by noninvasive methods (echocardiography). On the contrary, based on a presumable baroreflexinduced tachycardia, there might be an indirect positive inotropic effect. In addition, others have shown that PTHrP might exert inotropic effects mediated via coronary vasodilation (3). Finally, the most important concern is the lack of PTHrP concentration monitoring during the infusion, which would have demonstrated whether the responses were of pharmacological or of pathophysiological relevance. Indeed, in humoral hypercalcemia of malignancy, associated with increased PTHrP concentrations, no cardiovascular changes are systematically present. As congestive heart failure (CHF) is the most appropriate condition to demonstrate neurohumoral activation, we studied PTHrP plasma concentrations in this condition to establish its clinical relevance as a cardiovascular vasodilator, and we compared PTHrP concentrations with those of cardiac natriuretic peptides activated in CHF. Indeed, a colocalization of PTHrP with natriuretic peptides in myocytes secretory granules was previously suggested (4, 5). Thus, we obtained peripheral venous blood, after 30 min strict recumbency, from 10 healthy volunteers (CTR), 11 patients with angiographically-demonstrated coronary artery disease (CAD) and normal ejection fraction (EF), and 26 patients with congestive heart failure (CHF) in New York Heart Association (NYHA) class II (n 5 10; EF: 26 6 6%) and in NYHA class III-IV (n 5 16; EF: 19 6 5%). PTHrP (1– 84) was measured in whole plasma by an immunoradiometric assay (PTHrP IRMA 35400, Incstar, Stillwater, MN; sensitivity: 1.5 pmol/L; intra/interassay CV: 4/8%). Atrial natriuretic factor (CANF), the N-terminal part of its precursor (N-ANF), and brain natriuretic peptide (BNP) were measured on the same samples, after acetonitrile extraction on Sep-Pack C18-cartridges (Waters, Milford, MA) and using radioimmunoassays (Peninsula, Belmont, CA; intra/interassay CV: ,10% for the three natriuretic peptides). As expected in CTR, plasma PTHrP levels were less than 1.5 pmol/L; similarly undetectable values were obtained in all CAD and CHF patients. Positive samples (from patients with humoral hypercalcemia of malignancy), similarly handled and included in the same assay, were in the expected range (5–50 pmol/L) and demonstrated dilution curves parallel to standards. In contrast to PTHrP, plasma levels of the atrial (C- and N-ANF) and of the ventricular (BNP) peptides increased with the severity of cardiac disease. Median [range] plasma C-ANF were respectively 32[22– 69], 68[22– 130], 101[28–495], and 204[54–418] pg/mL in CTR, CAD, CHF I-II, and CHF III-IV patients (P , 0.0001 by Kruskal-Wallis test). N-ANF plasma concentrations were 333[158–762], 596[116–762], 726[434 –1926], and f

Received October 8, 1997. Address correspondence to: Professor Michel Rousseau, Division of Cardiology, University Hospital Saint Luc, Avenue Hippocrate 10/2800, B-1200 Brussels, Belgium.

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2023[806–4181] pg/mL (P , 0.0001), and BNP levels were 14[9–34], 29[13– 77], 66[23–121], and 201[37–336] pg/mL (P , 0.0001). Thus, in contrast to cardiac natriuretic peptides, the peripheral plasma levels of PTHrP do not increase in the presence of or with the severity of heart failure, at least in the range of sensitivity of presently available assays. Practically, this dismisses the potential interest of PTHrP as a clinically useful marker in CHF as well as its physiological or pathophysiological role in the control of systemic hemodynamics in man. Although a paracrine effect undetected by peripheral plasma levels cannot be ruled out, the actions of PTHrP reported by M. Wolzt et al. (1) might reflect a more pharmacological effect of PTHrP. Philippe L. Selvais, Julian E. Donckier, Laurent Rousseau, Sylvie A. Ahn, Jean-Marie Ketelslegers, and Michel F. Rousseau University Hospital Saint Luc Brussels, Belgium

References 1. Wolzt M, Schmetterer L, Dorner G, et al. 1997 Hemodynamic effects of parathyroid hormone-related peptide (1–34) in humans. J Clin Endocrinol Metab. 82:2548 –2551. 2. Nickols G, Nana AD, Nickols MA, Dipette DJ, Asimakis GK. 1989 Hypotension and cardiac stimulation due to the parathyroid hormone-related protein, humoral hypercalcemia of malignancy factor. Endocrinology. 125:834 – 841. 3. Ogino K, Burkhoff D, Bilezikian JP. 1995 The hemodynamic basis for the cardiac effects of parathyroid hormone (PTH) and PTH-related protein. Endocrinology. 136:3024 –3030. 4. Burton DW, Brandt DW, Deftos LJ. 1994 Parathyroid-hormone related protein in the cardiovascular system. Endocrinology. 135:253–261. 5. Deftos LJ, Burton DW, Brandt DW. 1993 Parathyroid hormone-like protein is a secretory product of atrial myocytes. J Clin Invest. 92:727–735.

Hemodynamic Effects of Parathyroid HormoneRelated Peptide: Is There A Pathophysiological Relevance?—Author’s Responseg To the editor: There is ample evidence for the suitability of noninvasive measurement of systolic time intervals to detect positive inotropic effects independent of reflectory changes in heart rate (1, 2). The method is easy to handle, has a very high sensitivity and reproducibility, and has a high signal-to-noise ratio. These advantages favor this technique for the characterization of systolic inotropic drug effects compared with echocardiography, which has a significantly higher short-term and day-to-day variability and is susceptible to observer-related influences (3). We consider it therefore unlikely that we have missed a major (direct or indirect) positive inotropic effect in our experiments. We nevertheless agree with the comment that the peripheral vasodilating effect of PTHrP deserves further studies. Our experiments were based on in vivo animal data and were designed to investigate short-term hemodynamic effects of PTHrP. It is apparent that the doses used in our experiments predominantly reflect pharmacological effects of PTHrP. Studies that could resolve the question raised by Selvais et al. (4), whether increased paracrine PTHrP activity plays a pathophysiological and/or hemodynamic role, will have to await the availability of specific inhibitors of PTHrP generation or receptor antagonists. Until then, in vivo studies are limited to exogenous administration of the peptide. Selvais et al., in the letter above, clearly show that measurement of PTHrP in peripheral blood fails to identify patients with coronary artery disease or congestive heart failure. Although stimulated PTHrP expression has been demonstrated in the presence of endothelin or norepinephrine (4), these results again underline the limitations of extrapolation of in vitro experiments to patients with activated neurohumoral conditions. We nevertheless agree that the potential paracrine role of increased PTHrP expression cannot be assessed from measurement of PTHrP levels in peripheral blood. g

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LETTERS TO THE EDITOR

Received December 9, 1997. Address correspondence to: Michael Woltz, M.D., Clinical Pharmacology, Allgemeines Krankenhaus Wien, Vienna University, Vienna, Austria A-1090.

Michael Wolzt, MD Department of Clinical Pharmacology, Vienna University Vienna, Austria A-1090

References 1. Belz GG. 1995 Systolic time intervals: a method to assess cardiovascular drug effects in humans. Eur J Clin Invest. 25 (Suppl 1):35– 41. 2. Wolzt M, Schmetterer L, Rheinberger A, et al. 1995 Comparison of noninvasive methods for the assessment of haemodynamic drug effects in healthy male and female volunteers: sex differences in cardiovascular responsiveness. Br J Clin Pharmacol. 39:347–359. 3. de Mey C, Belz GG, Nixdorf U, et al. 1992 Relative sensitivity of four noninvasive methods in assessing systolic cardiovascular effects of isoproterenol in healthy volunteers. Clin Pharmacol Ther. 52:609 – 619. 4. Hongo T, Kupfer J, Enomoto H, et al. 1991 Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation. J Clin Invest. 88:1841–1847.

Biochemical Markers of Bone Turnover for Predicting BMD in Early Postmenopausal Womenh To the editor: We have noted some disturbing inconsistencies and inaccuracies and found the breadth of analysis lacking in the recent report by Rosen et al. (1). The authors compared the responses of various biochemical markers of bone metabolism in a study of hormone replacement therapy (HRT) in postmenopausal women. The study reported results for N-telopeptides of type I collagen (NTx), deoxypyridinoline (Dpd), osteocalcin (OC), and bone alkaline phosphatase (BAP) in 236 women randomized to receive either calcium alone (CTL) or calcium plus HRT. The study was conducted over the course of one year, and bone mineral density of the lumbar spine and hip were measured by dual-energy x-ray absorptiometry at baseline, 3, 6, and 12 months. Markers were measured at baseline, 1, 3, 6, and 12 months. Almost all of the analyses had been previously reported by Chesnut et al. (2), with the exception of the addition of the Dpd measurements. The authors reported in the Results section of their paper that a significant decrease from baseline (P , 0.0001) was seen in the HRT group at 1 month with NTx, Dpd, and OC. However, in the Discussion, they stated that the earliest and most significant change from baseline was noted for NTx and OC. These two statements do not appear compatible in view of the statistics presented. We are concerned that this conclusion was drawn on the basis of the relative percent changes observed for the different markers, 228%, 210%, and 215%, respectively. Percent change, however, is not a statistical measure and ignores the contribution of the variability inherent in each of the different biochemical markers. Rosen et al. (1) reported a lack of predictive value of baseline Dpd for HRT-induced increases in spine BMD on the basis of the fact that there was no difference between first and fourth quartiles of the marker. However, third quartile Dpd values were considerably higher than those observed in all other quartiles, and fourth quartile results were higher than first and second. This suggests the possibility that above-median Dpd values might be associated with significantly higher spine BMD changes than below-median Dpd values, and possibly that the trend of increasing quartiles might also be significant. If either of these were so, the predictive value, or lack thereof, would depend upon the method of analysis and would be open to interpretation. In their receiver operating characteristic (ROC) analysis, the authors presented data for the change in marker levels at 6 months compared with one year BMD change. They described in Results that NTx provided a greater discrimination between gain and loss of BMD than the other markers. However, the authors report no statistical comparison of the areas under the ROC curves that leads to this conclusion. NTx, Dpd, and OC were reported to decrease significantly at 1 and 12 months, and presumably at 3 and 6 months (not stated) as the magnitudes of percent change are greater at 3 and 6 than at 1 month, with comparable standard error. However, the authors did not offer any comparisons using ROC analysis at those time points other than 6 months. h Received October 23, 1997. Address correspondence to: Claude D. Arnaud, Departments of Medicine and Physiology, University of California, San Francisco, 1710 Scott Street, Third Floor, San Francisco, California 94115.

LETTERS TO THE EDITOR The analysis of marker values in CTL was misleading as the authors chose to average all on-therapy values for their analysis rather than determine predictive value of baseline measurements, as was done for the HRT group. As that would be a desirable finding, we conclude that there was no baseline predictive value of the markers, at least for NTx, the commercial product of the study’s corporate sponsor. Thus, the reported data offers no insight on the ability of any of the 4 markers to predict BMD changes over 1 yr in postmenopausal women receiving calcium. As with spine BMD, hip BMD was reported to be increased significantly at 12 months in the HRT group [and decreased in the CTL group in the other published report of this study (2)]. However, no associations between any of the biochemical variables and hip BMD were reported. We presume there were none, or at least none favorable to the sponsor’s product. In their Discussion, Rosen et al. reported that a 20 –25% CV for NTx and Dpd in an individual is not unexpected and is similar to reports in other studies. The studies cited to support this statement make no report of intraindividual variability of Dpd measured by the assay they used. In fact, there are no published reports of intraindividual variability as high as the authors have stated for the Dpd assay; intraindividual CV for the Dpd assay used is reported to average between 10 –16% (3, 4). This lower figure affects the conclusions the authors have drawn regarding the relative balance between treatment response and variability for the two markers. In summary, we are concerned about the reporting of data and review of the literature by Rosen et al. and by the imprecise conclusions they have made in their paper. Their report fails to fully elucidate associations that exist between the various markers and BMD in HRT- or calciumtreated postmenopausal women. Such information is needed to enable physicians to appropriately select tools to aid in patient management. Claude D. Arnaud Departments of Medicine and Physiology University of California, San Francisco San Francisco, California 94115 Dean K. Jenkins Metra Biosystems, Inc. Mountain View, California 94043

References 1. Rosen CJ, Chesnut CH III, Mallinak NJS. 1997 The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab. 82:1904 –1910. 2. Chesnut CH III, Bell NH, Clark G, et al. 1997 Hormone replacement therapy in postmenopausal women: urinary N-telopeptide of type I collagen monitors therapeutic effect and predicts response of bone mineral density. Am J Med. 102:29 –37. 3. Popp-Snijders C, Lips P, Netelenbos JC. 1996 Intra-individual variation in bone resorption markers in urine. Ann Clin Biochem. 33:347–348. 4. Ju H-SJ, Leung S, Brown B, et al. 1997 Comparison of analytical and biological variability of three bone resorption assays. Clin Chem. 43:1570 –1576.

The Predictive Value of Biochemical Markers of Bone Turnover for Bone Mineral Density In Early Postmenopausal Women Treated with Hormone Replacement or Calcium Supplementation—Author’s Responsei To the editor: In response to the letter submitted above, we take issue with several comments about the article by Rosen et al. (1). First, we have published two manuscripts that report information from this randomized longitudinal trial examining the role of biochemical markers in predicting skeletal response to calcium supplementation vs. hormone replacement therapy (HRT) in early postmenopausal women. In the first paper, Chestnut and my colleagues report primarily on study design and the a priori primary outcomes measures: urinary N-telopeptide (NTx) and bone mineral density (BMD). The overwhelming majority of data presented in that paper centered on NTx, with virtually no additional information about other markers; in fact there was only one table (Table II) containing i

Received November 26, 1997. Address correspondence to: Clifford J. Rosen, M.D., Maine Center for Osteoporosis Research & Education, St. Joseph’s Hospital, 360 Broadway, Bangor, Maine 04401.

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correlation coefficients and ROCs, and a short paragraph in the Results section in respect to the other turnover indices. Complete data sets on free deoxypyridinoline (fDpd) were not available when the first manuscript was prepared and therefore could not be included in the original paper. Indeed, there was an overwhelming amount of data beyond the primary outcome measures, which necessitated, as customary in large multicenter trials, a second manuscript (2), comparing performance of other available biochemical markers, and their utility in predicting skeletal response to calcium or HRT. Second, concern was raised by the authors of this letter as to the comparison between changes in NTx and other markers after the first month of HRT, especially in respect to inherent variability in measures. The use of the term “earliest and most significant” is a qualitative descriptor of the change in NTx and osteocalcin. These indices had the most rapid and significant response to HRT (P , 0.0001) after 1 month. Discussion of variability on the ability to measure a change is found in the Discussion section as noted: “. . . however, when subjects’ coefficients of variation were compared to the percent change in the markers after the initiation of HRT, the mean change in NTx due to therapy was always greater than the biological variability, even at the early time points. This was not the case for fDpd. As an example, after 1 month of HRT, the mean percent change in NTx was 228% compared with the mean change in fDpd of 210%. After 6 months, the mean change in NTx was 242% compared with that in fDpd, which was 222%”.(1) Third, issues were raised about comparing 1st and 4th quartiles for fDpd. We could not find the reason why HRT subjects in the 3rd quartile of fDpd at baseline had a greater percent change in BMD after 1 year than those with higher fDpd in the 4th quartile. The data stand as analyzed several different times and as reported in the manuscript. We chose to use quartiles rather than medians because the goal of the analysis was to provide relevant statistics for an individual patient rather than an entire group. Indeed, this is a standard method of representing data from epidemiologic studies. Moreover, odds ratio analysis provides further evidence of the marker’s ability to predict gain in BMD if on HRT, or loss if not on HRT. Fourth, concern was raised about ROC analysis. Providing an ROC curve at all time points would not provide additive information and would introduce problems inherent in multiple testing. The 6th month time point was chosen because it was the time when the resorption markers were closest to their nadir, the latest time point before the end of the study, and formation markers had decreased by that time point. Fifth, there was some question about the “control” or calcium only group. The control group was included in the study as a control. The study was not designed to compare two intervention groups, calcium supplementation vs. HRT. As Chesnut et al. (2) noted, markers of bone remodeling did not change significantly in the control group throughout the study (average: 23.0% P 5 0.20). Therefore we chose to represent marker values as a mean over the entire study. Sixth, the authors inquire about use of other skeletal sites. The purpose of this paper was not to compare changes at the spine vs. those at the hip, but rather to compare markers of bone turnover. Chesnut provided sufficient femoral neck BMD to demonstrate therapeutic skeletal responsiveness. As reported previously, HRT has a much greater and more rapid effect on spine than hip BMD, so we chose a priori to examine lumbar BMD responsiveness to HRT (2). This choice of site also fits with ongoing clinical concerns related to changes in spine BMD after prolonged HRT. Seventh, the issue of percent C.V. is raised. The letter to the editor points out that the published within-patient C.V. for fDpd is 10 –16%. However, this does not affect the conclusions that the within-patient variability in fDpd is too great in this study to see a significant change before 6 months, at which time the average percent change due to therapy is only slightly greater than the C.V. Clifford J. Rosen, MD Maine Center for Osteoporosis Research & Education Bangor, Maine 04401

References 1. Rosen CJ, Chesnut II CH, Mallinak NJS. 1997 The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab. 82:1904 –1910.

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JCE & M • 1998 Vol 83 • No 3

LETTERS TO THE EDITOR

2. Chesnut II CH, Bell NH, Clark GS, et al. 1997 Hormone replacement therapy in postmenopausal women: urinary N-telopeptide of Type I collagen; monitoring therapeutic effect and predicting response of bone mineral density. Am J Med. 102:29 –37.

Linkage Analysis of Familial Hyperaldosteronism Type II—Absence of Linkage to the Gene Encoding the Angiotensin II Receptor Type 1j To the editor: Recently, Davies et al. (1) reported that somatic mutations of the coding region of the angiotensin II type 1 receptor (AT1) gene were not found in 17 aldosterone producing adenomas (APA) studied by single strand conformation polymorphism (SSCP) and direct sequencing, although other genetic defects of the 59 and 39 regulatory regions of the gene were not excluded by this study. A previous study showed no abnormalities, on SSCP analysis of the coding region of the AT1 gene, in angiotensin II responsive and angiotensin II unresponsive APAs (2). Recently, we have performed linkage analysis with a polymorphism located within 15kb of the AT1 gene in a 29-member family that included 7 individuals affected with familial hyperaldosteronism type II (FH II). FH II is characterized by primary hyperaldosteronism, dominant transmission, nonsuppressibility of aldosterone by dexamethasone, and absence of the hybrid CYP11B1/CYP11B2 gene known to be the cause of glucocorticoid remediable hyperaldosteronism (or FH I) (3, 4). Angiotensin II stimulates aldosterone production through activation of the G-protein coupled 7 transmembrane domain receptor, AT1. Like Davies et al., we hypothesized that a germ line mutation causing constitutive activation of the AT1 receptor could be the cause of FH II. Genomic DNA from 29 family members, including all 7 affected individuals, was extracted from blood leukocytes, and PCR amplification of a marker containing a dinucleotide (CA) repeat polymorphism present in a 0.9 kb EcoRI fragment approximately 15 kb downstream from the 39 end of the coding sequence of the AT1 gene was performed (5). Linkage analysis for this marker revealed an LOD score of 22.24 at a recombination fraction of 0.01 (Table 1), excluding this gene as a cause of FH II in this family. Figure 1 demonstrates an informative recombination, where affected individuals 2 and 3 do not share an allele for this gene. Since a LOD score of less than 22 was achieved with this (Table 1) and other markers nearby (data not shown), mutations of AT1 do not appear to be the cause of familial hyperaldosteronism type II. David J. Torpy and Constantine A. Stratakis Developmental Endocrinology Branch, NICHD, National Institutes of Health Bethesda, Maryland 20892-1862 Richard D. Gordon University of Queensland Department of Medicine, Greenslopes Hospital Brisbane, Queensland, 4120 Australia

FIG. 1. A segment of the 29 member pedigree with familial hyperaldosteronism type II. Genomic DNA was extracted from peripheral blood leukocytes and PCR amplification of a CA repeat polymorphic marker close to the angiotensin II receptor type 1 was performed, one primer was radiolabelled (g-32P) with T4 kinase. The PCR products were electrophoresed on a 6% polyacrylamide denaturing gel, the dried gel was exposed to X-ray film for 12–16 h. Individual 2 (alleles a/b) and individual 3 (alleles c/c) have the disease but do not share an allele for this marker for the AT1 gene. Individual 4 is of uncertain affectation status. The affected mother has the blc genotype.

References 1. Davies E, Bonnardeaux A, Plouin P-F, Corvol P, Clauser E. 1997 Somatic mutations of the angiotensin II (AT1) receptor gene are not present in aldosterone-producing adenoma. J Clin Endocrinol Metab. 82:611– 615. 2. Klemm SA, Ballantine DM, Tunny TJ, Stowasser M, Gordon RD. 1995 PCR-SSCP analysis of the angiotensin II type 1 receptor gene in patients with aldosterone-producing adenomas. Clin Exp Pharmacol Physiol. 22:457– 459. 3. Stowasser M, Gordon RD, Tunny TJ, Klemm SA, Finn WL, Krek AL. 1992 Familial hyperaldosteronism type II: five families with a new variety of primary hyperaldosteronism. Clin Exp Pharmacol Physiol. 19:319 –322. 4. Gordon RD, Stowasser M, Klemm SA, Tunny TJ. 1995 Primary hyperaldosteronism—some genetic, morphological, and biochemical aspects of subtypes. Steroids. 60:35– 41. 5. Davies E, Bonnardeaux A, Lathrop GM, Corvol P, Clauser E, Soubrier F. 1994 Angiotensin II (type 1) receptor locus: CA repeat polymorphism and genetic mapping. Hum Mol Genet. 3:838.

TABLE 1. LOD scores at different recombination fractions (U) for a microsatellite marker for the angiotensin II receptor type 1 gene analyzed in a family with seven members affected with familial hyperaldosteronism type II Marker

AT1

0

0.01

0.02

0.03

2`

22.24

21.65

21.3

LOD score at U 5 0.04

21.07

0.05

0.1

0.2

0.3

20.89

20.37

0.032

0.14

j

Received August 7, 1997. Address correspondence to: David J. Torpy, Section on Pediatric Endocrinology, Developmental Endocrinology, Branch, NICHD, 9000 Rockville Pike, Bethesda, Maryland 20892-1862. The Journal of Clinical Endocrinology and Metabolism invites the submission of Letters to the Editor. These should not be substitutes for Rapid Communications, but should be written for the purpose of scholarly commentary or protest. Please send such letters to Maria I. New, MD, Editor-in-Chief, at the address given on the Author and Subscription Information page at the front of each issue.