417

Acid-induced Osteoporosis: An Experimental Model of Human Osteoporosis

U.S. BARZEL

SUMMARY
Adult animals, male or female, react to ammonium chloride ingestion with a non-hormonal, generalized, slow but progressive and unrelenting mobilization of bone and develop osteoporosis which seems indistinguishable in all parameters measured from human osteoporosis. (Table I).
Table L

Comparison of Human Osteoporosis and Experimental (Acid-induced) Osteoporosis. Parameter Blood calcium & phosphorus Bone histology Bone microradiography Bone per cent ash Bone calcium content Bone crystallography Human osteoporosis normal osteoporosis increased resorption normal formation normal diminished normal Animal model normal osteoporosis increased resorption normal formation normal diminished normal

Osteoporosis can be defined as a condition in which generalized, progressive diminution in bone density renders bones increasingly vulnerable to fracture. Clinically, osteoporosis has a slow natural history and an unpredictable course: it is virtually impossible to predict if and when any given patient is about to have a fracture (21), and in some cases the progression of the disease seems to have been arrested altogether (19). This slow and unpredictable nature o f osteoporosis makes it desirable to use experimental animal models in its study. The present paper will
Metabolic Endocrine Laboratory, Department of Medicine, Montefiore Hospital and Medical Center, Albert Einstein College of Medicine, Bronx, New York.

418 summarize available information on the model of acid-induced osteoporosis (12, 3, 4, 5, 6).

THEORETICAL CONSIDERATIONS AND MECHANISMS Since the end of the last century, bone has been recognized to contain large amounts of alkaline salts and has been thought likely to participate in the maintenance of acid base balance of the organism. The fact that during acid loading urinary calcium excretion always increases gave support to this idea (2). Recent studies in normal man (14, 15), in renal acidosis (10, 16), and in starvation acidosis (18) gave additional support to the concept that bone was mobilized in acid loading and its salts made available to augment the buffering capacity of the extracellular fluid. In 1969 we suggested, on the basis of experimental observations, that cellular mechanisms involved with bone formation and bone resorption were responsive to changes in acid base balance" microradiographic studies demonstrated that the chronic ingestion of ammonium chloride caused an increase in bone resorption surfaces without change in bone formation surfaces, and, conversely, the chronic ingestion of an alkali solution caused an increase in bone formation surfaces without change in bone resorption surfaces (4). Delling and Donath (9) demonstrated by electronmicroscopy that the bone resorption which occurs as a direct consequence of the dietary ingestion of excess acid is indeed mediated by bone cells. They demonstrated that osteoclasts and osteocytes were activated in acid loaded animals even in the absence of the parathyroid glands. Acid-induced bone resorption was shown by Reed and Beck to be independent of gastrointestinal and renal factors and was observed in animals after nephrectomy and after removal of the entire GI tract (17). Cuisinier-Gleizes et al. also found that ammonium chloride ingestion caused bone resorption in the presence and in the absence of the parathyroid glands (8). This group, however, did not find histologic evidence of cellular mediation of this response. They believe that the resorption of bone is not mediated by cells and also that bone formation is depressed in ammonium chloride fed animals. The observations cited above further strengthen the hypotesis that resorption of bone is a bona fide physiologic compensatory response to the ingestion of acid.

419 THE EXPERIMENTAL MODEL Adult animals given ammonium chloride solution as their drinking fluid, or fed ammonium chloride in the diet, develop histologic osteoporosis (12, 4, 5, 9, 8), with a ratio of osteoblasts to osteoclasts comparable to that of control animals (8). Ammonium chloride ingestion does not affect the length or volume of the femur (4, 5, 6). There is a decrease in femoral density as measured gravimetrically, by Archimedes' principle (4, 5, 6), or by radiograph densitometry. These density measurements correlate closely with each other and with chemical analyses (7). The latter show that bone fat free weight, bone ash, bone matrix, and the calcium content of the femur all decrease as a result of ammonium chloride ingestion* but the ratio of ash to matrix and the relationship of fat free weight to calcium content of the bone remain unchanged (4, 5, 6). (Figs. 1 and 2). Chrystallographically, the size of the appatite crystals and the per cent of crystalline and amorphous fractions are not affected by ammonium chloride ingestion (5). Other observations made in animals chronically fed ammonium chloride reveal that blood calcium and phosphorus are normal (4). Blood sodium and potassium are also normal (4), and osteoporosis develops in the presence of normal arterial blood pH (4, 8). The blood chloride tends to be high and the blood carbon dioxide content slightly depressed (4). Clearly, the organism makes available stored base buffer, the bone carbonate and the hydroxyappatite, to help maintain acid base neutrality in the face of excessive ingestion of acid. Observations are essentially the same when male or female rats are studied (4, 5, 6). The degree of osteoporosis that is observed seems to depend on the dose of ammonium chloride used and the duration of its administration. Low calcium diet has been shown previously to produce osteoporosis by a parathyroid dependent mechanism (11, 13). The effect of ammonium chloride is evident in animals given a low calcium diet to the same degree as in animals given a regular laboratory diet. The combination of ammonium chloride and a low calcium diet is additive in its effect, causing a more profound degree of osteoporosis than either ammonium or a low calcium diet alone (4, 5). (Figs. 1 and 2). Another potential mechanism for the development of osteoporosis is the withdrawal of estrogenic hormones (1). The effect of ammonium

420
300 - O=REGULAR DIET,Distilledwater 9 =LChVCALCIUMDIET,1.5%NH4CI ~ 250-p ~ y

@~A A 9

200--

150-u 10C-y = 0.2146 x +3.463

r= 0.88
5G - p= < 0.0001

10o 600 I 700 l 800 I 900 I 1000 I 1100 I 1200 I 1300 I 1400 l 1500 I
FAT FREE SOLID (mg)

Figure 1. F a t free solid w e i g h t a n d c a l c i u m c o n t e n t o f t h e r i g h t f e m u r o f 2 5 0 g male

rats f e d a regular or a l o w c a l c i u m diet a n d distilled w a t e r or 1.5% a m m o n i u m c h l o r i d e f o r 3 3 0 days. (Modified f r o m Barzel & J o w s e y (4)).

o REGULAR DIET, DISTILLED WATER '~ REGULAR DIET, 2% NH4CI 9 LOW CALCIUM DIET, DISTILLED WATER 9 LOW CALCIUM DIET, 2% NH4CI
300"

CALCIUM mg

2509 ~

oo~
6 o

zOO-

150-

~I

9
I I

9 Z~Z~1.414.6X-415.7 r 9 0.78 p < 0.0001

I I I

100

LSO0

1.400 I.,500 DENSITY, gm/crrr ~

1,600

Figure 2. D e n s i t y a n d c a l c i u m c o n t e n t o f t h e r i g h t f e m u r o f 5 5 0 g m a l e rats fed a regular o r a low c a l c i u m diet a n d distilled w a t e r or 2% a m m o n i u m c h l o r i d e for 6 m o n t h s . ( M o d i f i e d f r o m Barzel (6))-

421 chloride was c o m p a r e d with t h a t o f o o p h o r e c t o m y in a d u l t female rats. O o p h o r e c t o m i z e d animals, a l l o w e d a regular diet ad lib., failed t o develop a n y c h a n g e in b o n e density, fat free weight, ash a n d c a l c i u m c o n t e n t o f the f e m u r , e x c e p t w h e n given a m m o n i u m chloride. T h e o o p h o r e c t o m i z e d animals were n o t m o r e sensitive t o a m m o n i u m chloride t h a n c o n t r o l animals (6).

REFERENCES 1. Aitken, J.M., Armstrong, E. & Anderson, J.B.: Osteoporosis in the mature female rat and the effect of oestroge and/or progesterone replacement therapy in its prevention. J. Endocr. 55, 79--87 (1972) 2. Albright, F.A. & Reifenstein, E.C.: The Parathyroid Gland and Metabolic Bone Disease: Selected Studies, Williams and Wilkins, Baltimore, p. 241, 1948 3. Barzel, U.S.: Acid base balance and bone metabolism: A study in the etiology of osteoporosis. Clin. Res. 14, 485 (1966) 4. Barzel, U.S. & Jowsey, J.: The effect of chronic acid and alkali administration on bone turnover in adult rats. Clin. ScL 36, 517-524 (1969) 5. Barzel, U.S.: The effect of excessive acid feeding on bone. Calc. Tiss. Res. 4, 94--100 (1969) 6. Barzel, U.S.: Studies in osteoporosis: The long term effect of oophorectomy and of ammonium chloride ingestion on the bone of mature rats. Endocrinology 96, 1304--1306 (1975) 7. Colbert, C. & Barzel, U.S.: Radiographic determination of rat bone mineral loss in dietary-induced osteopoxosis. Invest. RadioL 7, 339--340 (1972) 8. Cuisinier-Gleizes, P., Behest, D., George, A. & Thomasset, M.: Thyroparathyroid glands, bone and acid-base homeostasis. XIth Europ. Symp. Calc. Tiss., May 25--29, Elsinore, Denmark, Abstract book, p. 70, 1975 9. Delling, G. & Donath, K.: Morphometrische, electronen-mikroskopische und physikalisch-chemische Untersuchungen uber die experimentelle Osteoporose bei chronischer Acidose. Virchows Arch. Path. Anat. Abt. A 358, 321--330 (1973) 10. Goodman, A.D., Lemann, J. Jr., Lennon, E.J. & Relman, A.S.: Production, excretion, and net balance of fixed acid in patients with renal disease. ]. clin. Invest. 44, 495--506 (1965) 11. Harrison, M. & Fraser, R.: Bone structure and metabolism in calcium-deficient rats. J. Endocr. 21, 197-204 (1960) 12. Jaffe, H.L., Bodansky, A. & Chandler, J.P.: Ammonium chloride decalcification as modified by calcium intake: relation between generalized osteoporosis and ostitis fibrosa. ]. exp. Med. 56, 823--834 (1932) 13. Jowsey, J. & Raisz, L.G.: Experimental osteoporosis and parathyroid activity. Endocrinology 82, 384--396 (1968) 14. Lemann, J. Jr., Lennon, E.J., Goodman, A.D., Litzow, J.R. & Relman, A.S.: The net balance of acid in subjects given large loads of acid or alkali. J. clin. Invest. 44, 507--517 (1965) 15. Lemann, J. Jr., Litzow, J.R. & Lennon, E.J.: The effects of chronic acid loads in normal man: Further evidence for the participation of bone mineral in the defense against chronic metabolic acidosis. J. clin. Invest. 45, 1608--1614 (1966)

422 16. Litzow, J.R., Lemann, J. Jr., & Lennon, E.J.: The effect of treatment of acidosis on calcium balance in patients with chronic azotemic renal disease. ]. clin. Invest. 46, 2 8 0 - 2 8 6 (1967) 17. Reed, S.W. & Beck, N.: Acute metabolic acidosis on calcium mobilization from bone and calcium excretion, and on parathyroid hormone actions. Clin. Res. 23, 328 A (1975) 18. Reidenberg, M.M., Haag, B.L., Channick, B.J., Shuman, C.R. & Wilson, T.G.G.: The response of bone to metabolic acidosis in man. Metabolism 15, 2 3 6 - 2 4 1 (1966) 19. Schenk, M., Rodstein, M. & Barzel, U.S.: Frequency and rate of progression of osteoporosis. Israel J. Med. Sci. 10, 1471 (1974) 20. Smith, D.A., Lindsay, R., Boddy, K., Elliott, A. & A n d e r s o n , J . : The use of total b o d y in vivo neutron activation analysis in balance studies in rodents. 21. Urist, M.R., Gurvey, M.S. & Fareed, D.O.: Long term observations on aged women with pathologic osteoporosis. In: Osteoporosis, Barzel, U.S. (ed.), Grune and Stratton, New York, pp. 3--37, 1970