Bone changes in alcoholic cirrhosis of the liver

Path. Res. Pract. 184, 480-485 (1989)

Bone Changes in Alcoholic Cirrhosis of the Liver A Histomorphometric Study D. Chappard, B. Plantard, H. Fraisse, S. Palle, C. Alexandre and G. Riffat LBTO - Laboratoire de Biologie du Tissu Osseux Faculte de Medecine, Saint Etienne, France

SUMMARY The effects of alcoholic liver cirrhosis on the calcium, calciotropic hormones (parathyroid hormone and 25 hydroxy vitamin D) and testosterone were examined in twenty men. A transiliac bone biopsy was performed in each patient. A double tetracycline labeling was done in 17 patients. Radiological signs of osteoporosis (crushed vertebrae and rib fractures) were rarely observed but a low trabecular bone volume (BV/TV) was consistently encountered (BV/TV = 14.2 ± 4.6; normal 18.8 ± 4.8; p < .001). Osteomalacic signs were never observed in spite of very low calcium and 25 hydroxy vitamin D levels in the blood. Increased osteoclastic eroded surfaces (ES/BS = 8.1 ± 5.2; normal 3.7 ± 1.1; p < .001) were not related with an hyperparathyroidism but a significant correlation was found with testosterone levels (r = .45; P < .05). Histodynamic measurements indicate a drastically decreased osteoblastic activity with a very low bone formation rate (BFR/BS = .009 ± .001; normal .0175 ± .0125; P < .001). This could explain the net increase of eroded surfaces and that osteomalacia cannot be observed even in the presence of profound calcium and vitamin D changes in alcoholics.

Introduction Chronic ethyl alcohol consumption is associated with an increased risk of bone fractures, four times more common than in normal subjects. Fractures are related to trauma, occurring more frequently during drunkness, as well as an underlying bone fragility. Both osteomalacia and osteoporosis have been reported in chronic liver diseases13, 19. Disturbances in calcium metabolism and calciotropic hormones (parathyroid hormone and vitamin D) are now well recognized in chronic alcoholism 1, 10. However, the relationships between these metabolic modifications and the bone changes at both the tissue and the cellular level have received little attention. Furthermore, alcoholic patients do not represent a sharply defined group and the importance and duration of alcoholic abuse are difficult to establish clinically. Patients with a decompen0344·0338/89/0184·0480$3.5010

sated alcoholic cirrhosis represent a more homogeneous group in which metabolic and bone changes are presumably at the upmost. The purpose of the present investigation was to study metabolical and bone changes in a group of patients with a decompensated cirrhosis. Bone morphometry, a powerful tool in the study of metabolic bone diseases, was used.

Material and Methods We have studied twenty men (mean age: 58.6 ± 10.6 y.o.) with a decompensated alcoholic cirrhosis of the liver from the GastroEnterology Unit. No patient had a previous history of corticosteroid, fluoride or vitamin D intake and renal disease. In 16 out of them, the diagnosis was proven by peritoneoscopy and liver biopsy. In the other 4 patients, the diagnosis was ascertained on © 1989 by Gustav Fischer Verlag, Stuttgart

Bone in Alcoholic Cirrhosis . 481 Table 1. Biochemical results determined in 20 men with an alcoholic cirrhosis of the liver BLOOD CALCIUM Immollll n=20 IONIZED CALCIUM (mmollll n=20 BLOOD PHOSPHATE (mmollll n=20 SERUM ALBUMINE (olll n=20 25 OH VITAMINE D (mmollll n=18 PARATHYROID HORMONE (lUll) n=19 TESTOSTERONE (no/ml) n=17

CIRRHOTICS 2.10 ± .12

NORMAL VALUES 2.25 ± .12

COMPARISON p < .001

.98 ± .07

1.18 ± .06

p< .001

1.06 ± .12

1.15 ± .17

p < .01

30.9 ± 4.7

44.5 ± 2.7

p< .001

23.9 ± 21.3

58 ± 15

P< .001

3.6 ± 1.2

3±1

N.S.

5.25 ±1.1

3.14 ± 2.6

the basis of clinical findings and biological disturbances and confirmed in 3 cases at surgery for portal hypertension. The last patient had diffused nodules on the liver surface at autopsy. The severity of the cirrhosis was evaluated according to the classification of Child and Turcotte6• Five patients had a mild hepatocellular insufficiency (grade A), 14 had a moderate (grade B) and one a severe (grade C) insufficiency.

X ray examination Biconcave and fractured vertebrae were recognized on a lateral lumbar X ray film including the ten vertebral bodies T 8 through L5. The radiological examination was normal in 13 patients. Only two cirrhotics (with back pain) presented numerous vertebral compression deformities. Old rib fractures were detected on chest radiographs in only 3 patients.

Biochemical measurements The blood samples were analysed for clacium, ionized calcium, phosphate, albumine and total proteins on a Technicon® SMA II auto analyser. Both total and ionized calcium were decreased significantly in cirrhotic patients (Table 1). Significant low phosphate levels were regularly observed. Serum 25 hydroxy vitamin D (25-0H-D) levels were determined by radio competition. The mean serum 25-0H-D of the entire group of cirrhotics was signif-

p < .001

icantly decreased. Of these, 8 had serum 25-0H-D levels less than 10 mmolli. A carboxy terminal antiserum was used to measure the parathyroid hormone (PTH). All the serum PTH results were within the normal range. Serum PTH did not correlate with total calcium or ionized calcium. Blood testosterone was measured by a radio immunological method. Values were drastically decreased in 10 patients, one was borderline, the remainder being within the normal range.

Bone biopsy A morphometric analysis of a undecalcified transiliac bone biopsy was done in the 20 patients. A double tetracycline labeling with demethyl chlortetracycline (Ledermycine® daily dose 600 mg) was done in 17 cases. A 2 - 12 - 4 days labeling schedule was used. Bone biopsies were fixed and embedded in methacrylates according to previously described procedures2• Sections were cut dry using a lung Type K® microtome with HK 3 tungsten carbide knives. Four 15 J.lm sections for fluorescence study of tetracycline were mounted dry and unstained in NeoEntellan®. Eight 7 J.lm sections for histomorphometric measurements of bone and osteoid parameters were stained by free floating in the dye solutions. A modified Trichrome Goldner procedure was used. Measurements were done on semiautomatic and automatic image analyser sys-

Table 2. Histomorphometric results determined in 20 men with an alcoholic cirrhosis of the liver EOIIEVOLUME 1(%) BVITV OSTEOID VOLUME 1(%) OV/BV OSTEOID SURFACES 1(%) OS/BS OSlEOID THICKNESS Itum) O.Th MINERAL APPOSITION RAlE Ifum) MAR BONE FORMATION RAlE Itmm3/mm2lvearl BFR/BS WALL THICKNESS I(um) W.Th ERODED SURFACES 1(%) ES/BS

CIRRHOTICS 14.2 ± 4.6

NORMAL VALUES 18.8 ± 4.8

COMPARISON P < .001

1.5 ± 1.5

2.8 ± 1.8

P < .02

9.7 ± 8.5

15.5 ± 6.1

P < .02

5.3 ± 2.1

8.0 ± 4.4

P < .02

.3

.72±.12

P< .001

.009 ± .001

.0175 ± .0125

P< .001

39.6 ± 8.1

50.2 ± 8.7

P< .001

8.1 ± 5.2

3.7 ± 1.1

P< .001

± .15

482 . D. Chappard, B. Plantard, H. Fraisse, S. Palle, C. Alexandre and G. Riffat terns developed in this laboratory. The accuracy of these systems is better than .3%. The following histomorphometric parameters were determined: - Trabecular bone volume (BVrrv; fractional volume corresponding to the amount of the cancellous space occupied by trabecular bone) - Osteoid volume (OVIBV) and osteoid surfaces (OSIBS) - Osteoid thickness (O.Th; corresponding to the average width of the osteoid seams covering bone forming surfaces) - Mineral apposition rate (MAR; provided by UV light measurement of the double tetracycline label deposited by osteoblasts) - Bone formation rate, tissue level (BFRlBS; corresponding to the volumetric amount of bone elaborated by osteoblasts) - Mean wall thickness (W.Th; corresponding to the thickness of complete trabecular bone structural packets) - Eroded surfaces (ESIBS; corresponding to the amount of trabecular surfaces covered by scalloped osteoclastic lacunae) Abbrevations of morphometric parameters were used according to the recent nomenclature proposed by the American Society for Bone and Mineral Research. Data were compared with normal values from 19 men of similar age obtained with the same methodology by Meunier et a1. 15 • Comparisons were made using Student's t test. All values are expressed as mean ± SD.

Results

Bone histological findings The trabecular bone volume was significantly decreased (Fig. 1) in 13 patients when compared with age-matched

controls. They were below the spontaneous fracture threshold of 14%. The two patients with the radiological vertebral abnormalities appeared in this group. All the osteoid parameters were decreased in this patient series and the MAR was low in all of them. The osteoblastic bone formation rate was drastically reduced and was zero in 5 patients. The thickness of trabecular packets, W.Th was significantly decreased as a consequence of the low osteoblastic activity. Eroded surfaces were highly increased in 15 patients. A significant correlation (r = .45; P < .05) was observed between the resorption surfaces and the testosterone levels (Fig. 2). From a morphological point of view, the depth of the resorption surfaces seemed to be surprisingly low and composed of numerous minute single bites. Very thin osteoid seams incompletely filled some of these eroded surfaces (Fig. 3). Tunneling and hook resorption figures (frequently observed in hyperparathyroid states) were never observed. Discussion The present study has shown extensive skeletal osteoporosis at the histological level in this series of twenty men with a decompensated alcoholic cirrhosis. It is noteworthy that only two patients presented vertebral compression deformities (a radiological sign of advanced trabecular osteoporosis), and three had rib fractures (a site containing a larger amount of cortical bone). It has been

Fig. 1. Bone biopsy from a cirrhotic patient with low trabecular bone volume (not the rare trabeculae in the cancellous space; C = outer cortical). Modified Goldner's trichrome, x 40.

Bone in Alcoholic Cirrhosis . 483

demonstrated that alcoholism is related to a decreased bone mineral content and an increased prevalence of bone fractures 18 • Vertebral compression fractures, however, appear rarely in cirrhotics: 12 out of 19 patients in a series of Meunier et al. 16 and none in the 24 patients reported by Patri et a1. 19 • A high incidence of both osteomalacia and

osteoporosis was reported in patients with liver cirrhosis 13, 19. The hypocalcemia associated with low 25-0HD levels mimics the biological profile of osteomalacic patients. Hypocalcemia is a regular finding in cirrhotic patients and is linked to hypoalbuminemia due to hepatic disturbances 19 • Low vitamin D levels have been related to

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r = .45 P < .05 n =17

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Fig. 2. Correlation between testosterone blood level and total trabecular resorption surfaces in 17 men with an alcoholic cirrhosis of the liver.

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Fig. 3. A bone trabecula from a patient with alcoholic cirrhosis. Note the minute single bites on the upper eroded surface (black arrow) and the presence of a very thin layer of osteoid (white arrow) x 300.

484 . D. Chappard, B. Plantard, H. Fraisse, S. Palle, C. Alexandre and G. Riffat an impaired 25 hydroxylation in the cirrhotic liver ll • However, recent studies have shown that 25 hydroxylation is normal in cirrhotics l7 . The half life of the 25-0H-D was shown to be reduced in these patients 12 and inactive metabolites are produced20 but they could not be measured in the present study. Poor sunshine exposure is recognized as the main factor leading to depletion of vitamin D in cirrhotics because 25-0H-D levels rise during summer in alcoholic patients 10, 12. Absence of osteomalacia in our series is in accordance with other studies!6,17 and is not surprizing even in the presence of a profound hypocalcemia and hypovitaminosis D. A drastic decrease in the osteoblastic activity is observed4 as illustrated by the low values reached by osteoid parameters and mineral apposition rate. The thinning of trabecular bone packets (W.Th) confirms the defect in the osteoblastic activity!,17. The increased eroded surfaces observed in cirrhotic patients were first related to a secondary hyperparathyroidism!,16. The low serum ionized calcium levels observed in cirrhotics 14 was thought to be responsible for an increase in PTH secretion. Assays with carboxyterminal immunsera have regularly shown normal results 1,4, 13, 19. An assay with a N terminal antibody was reported to give high PTH values 14 associated with an increased urinary cAMP production. However, Darnis7 pointed out that hypomagnesemia observed in cirrhotics could induce an insensivity of the peripheral PTH receptors and a decreased hormone release by parathyroid glands. The increased eroded surfaces observed in cirrhotic patients could reflect the osteoblastic deficiency rather than a true hyperparathyroidism. The osteoblasts stop working before they have finished the elaboration of a complete bone packet. This situation leads to a thinning of the mean wall thickness and an apparent increase of resorption surfaces. The reduced osteoblastic activity was thought to be related to a shortened osteoblast life span l7 . A direct toxic effect of ethanol has been advocated. Fallon et al. 8 failed to demonstrate alcohol-induced ultrastructural changes in bone cells but ethanol could act directly to inhibit mitotic index and activity of bone cells9. Indirect depressive effect has also been advocated since numerous hormonal systems are altered by chronic ethanolism lO • In a previous study3, we have suggested a correlation between bone mass and circulating testosterone levels in alcoholic men which present clinical and biochemical signs of hypoandrogeny22. Patients with decreased sex hormones output from other causes also have bone osteoporosis associated with a low osteoblastic activity. Cirrhotics often present high blood cortisol concentrations (not measured in this study)2! and osteoblastic activity is decreased in the presence of hypercorticism. So several factors accumulate to decrease osteoblastic bone formation. Unfortunately, osteocalcin (serum GLA - protein, BGP), a useful biochemical marker of the osteoblastic activity was not measured at the beginning of the present study. Therefore eroded surfaces composed of minute single bites in light microscopy appeared incompletely filled by bony layers at the scanning electron microscopic leveP. The net increase of eroded surface extent also reflects the osteoblastic defect and the correlation found

between testosterone and resorption surfaces supports this idea. References 1 Bikle DD, Genant HK, Cann C, Recker RR, Halloran BP, Stewler GJ (1985) Bone disease in alcohol abuse. Amer Int Med 103: 42-48

2 Chappard D, Alexandre C, Camps M, Montheard JP, Riffat G (1983) Embedding iliac bone biopsies at low temperature using glycol and methyl methacrylates. Stain Technol 58: 299-308 3 Chappard D, Alexandre C, Fraisse H, Riffat G (1983) L'os du cirrhotique: relation entre la masse osseuse et la testosteronemie plasmatique. Presse Med 12: 524 4 Chappard D, Laurent JL, Fraisse H (1982) L'os du cirrhotique. Nouveau concept integrant biologie et histomorphometrie. Nouv Presse Med 11: 528-529 5 Chappard D, Plantard B, Alexandre C (1987) Evidence for a decreased osteoblastic activity in alcohol-induced osteoporosis in man: a histomorphometric and scanning electron microscopic study of bone biopsies - Abst -. In: Cohn DV et aI. (Eds.) Calcium Regulation and Bone Metabolism, vol 9. Excerpta Medica Amsterdam-New York-Oxford, pp 700 6 Child III CG, Turcotte JG (1964) Surgery and portal hypertension. In: Child III CG (Ed.) The Liver and Portal Hypertension. pp 50, W. B. Saunders, Philadelphia and London 7 Darnis F (1973) Hypocalcemie des cirrhoses et troubles du metabolisme hepatique de la vitamine D. Nouv Presse Med 2: 2443-2445 8 Fallon M, Baran DT, Craig RB, Teitelbaum SL (1981) Ultrastructure of the rat epiphyseal growth plate following chronic ethanol ingestion. Calcif Tissue Int 33: 381-384 9 Farley JR, Fitzsimmons R, Jorch UM, Lau KHW (1985) Direct effects of ethanol on bone resorption and formation in vitro. Arch Biochem Biophys 221: 305 10 Gascon-Barre M (1985) Influence of chronic ethanol consumption on the metabolism and action of vitamin D. J Amer Coli Nutr 4: 565-574 11 Hepner GW, Roginski M, Fai Moo H (1976) Abnormal vitamin D metabolism in patients with cirrhosis. Am J Dig Dis 21: 527-532 12 Jung RT, Davie M, Hunter JO, Chalmers TM, Lawson DEM (1978) Abnormal vitamin D metabolism in cirrhosis. Gut 19: 290-293 13 Long RG, Meinhard E, Skinner RK, Vargheze Z, Will MR, Sherlock S (1978) Clinical, biochemical and histological studies of osteomalacia, osteoporosis and parathyroid function in chronic liver diseases. Gut 19: 85-90 14 Louboutin JY, Feuillu A, Allain H, Bernat M, Meadeb J, Pawlotsky Y (1980) Hyperparathyroidie secondaire des cirrhoses; arguments apportes par Ie calcium ionise, I'AMP cyclique urinaire, la parathormonemie N terminale. Rev Rhum 47: 707-713 15 Meunier P, Courpron P (1976) Iliac trabecular bone volume in 236 controls. Representativeness of iliac samples. In: Jaworski ZFG (Ed.) Bone Morphometry. 1st Int. Workshop on Bone Morphometry. Ottawa University Press, pp 100-105 16 Meunier P, Dechavanne M, Prost G, Edouard C, BernardJ, Courpron P, Tolot F (1973) L'os des cirrhotiques etudie par lecture quantitative de la biopsie osseuse. Lyon Med 229: 1089-1095 17 Mobarahan SA, Russell RM, Recker RR, Posner DB, Iber FL, Miller P (1984) Metabolic bone disease in alcoholic cirrhosis: a comparison of the effects of vitamin D2, 25-hydroxyvitamin D or supportive treattnent. Hepatology 4: 266-273

Bone in Alcoholic Cirrhosis . 485 18 Nilsson BE, Westlin NE (1973) Changes in bone mass in alcoholics. Clin Orthop 90: 209 19 Patri B, Le Charpentier Y, Gueris J, Calmette C, Dubrisay J (1978) Vitamine D, parathormone, calcitonine et histomorphometrie osseuse chez les cirrhotiques. Rev Rhum 45: 631-640 20 Posner DB, Russel RM, Absood S, Connor TB, Davis C, Martin L, Williams JB, Norris AH, Merchant C (1978) Effective 25-hydroxylation of vitamin D2 in alcoholic cirrhosis. Gastroenterology 74: 866-870

21 Rico H, Gomez-Castresana F, Cabranes JA, Almoguera I, Lopez-Duran L, Matute JA (1985) Increased blood cortisol in alcoholic patients with aseptic necrosis of the femoral head. Calcif Tissue Int 37: 585-587 22 Van Thiel DH (1981) Hypothalamic - pituitary - gonadal function in liver disease. Prog Biochem Pharmacol18: 24-34

Received July 3, 1987 . Accepted in revised form September 9, 1988

Key words: Bone histomorphometry - Liver cirrhosis - Parathyroid hormone - Vitamin D - Alcoholic cirrhosis D. Chappard, LBTO - Laboratoire de Biologie du Tissu Osseux, Faculte de Medecine, 15 rue Ambroise Pare, 42023 Saint Etienne, Cedex 2, France