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Chronic renal failure and vitamin d metabolites: A status report
J. steroid Biochem. Vol. 19, No. I, pp. 517-523. Printed in Great Britain. All rights reserved
1983 Copyright
0022-473 l/83 $3.00 + 0.00 0 1983 Pergamon Press Ltd
CHRONIC RENAL FAILURE AND VITAMIN D METABOLITES: A STATUS REPORT CLAUSCHR~~TIANSEN Department of Clinical Chemistry, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark
SUMMARY Seventeen undialysed adult patients with chronic renal failure took part in a controlled study of the effects of 1,25(OH),D, and D3. After a 6-month observation period the patients were allocated at random to 2 groups for 6 months of treatment with either 1,25(OH),D, (mean dose 0.5 pg daily) or D3 (dose 1OOpgdaily). Treatment was then discontinued and the patients were studied for a further 3 months. The patients as a group initially had a moderate renal osteodystrophy. During treatment serum iPTH decreased in both groups but most markedly in the 1,25(OH)2Dogroup in which the iPTH values became normal. Serum creatinine increased during treatment in both groups, in the group receiving 1,25(OH),D, this was coupled with an increase in serum calcium to within the normal range. Our data demonstrate that 1,25(OH),Ds treatment in patients with chronic renal failure leads to a further reduction in renal function, which may be partially reversible. Physicians should therefore be reluctant to give vitamin D analogues to patients with chronic renal failure unless they have severe symptomatic renal osteodystrophy.
METHODS
INTRODUCTION Decreased or absent la-hydroxylating activity is seen in progressive chronic renal failure (CRF) [l, 21, and is believed to be important in the development of renal osteodystrophy [3,4]. A number of uncontrolled studies have been published concerning the effects of the vitamin D analogues la-hydroxycholecalciferol (la-OHD,) and 1,25-dihydroxycholecalciferal (1,25(OH),D,) on various aspects of calcium and phosphorus metabolism, including a number of observations on the effect on bone histology [SS]. Most of the reports are positive, some even enthusiastic. Hypercalcemia is a well-known complication to therapy, and deterioration of renal function in CRF has been suggested in a controlled study of la-0HD3 in predialysis patients [9]. We have compared the effects of 1,25(OH)*D3 and vitamin D, in a controlled study of predialysis CRF patients with special reference to a possible effect on both bone and biochemical parameters. as well as renal function.
PATIENTS The study comprised of 17 patients with chronic renal failure. They were 5 men and 12 women aged 26-70 yr (mean age 53 yr). Renal function had been relatively stable for more than 1 yr and the creatinine clearance. was between 5 and 35 ml/min. Details of the selection criteria have been given elsewhere [lo].
Design of study
After the preliminary investigations (t-a) the patients were studied for 6 months (until to) to determine the spontaneous course in the observed parameters. The 17 patients were randomly allocated into two groups: one was treated with 1,25(OH)*D3, and the other with vitamin D, (see below). During the following 6 months (to-Q all patients received 0.5 g calcium daily. Patients in the 1,25(OH)*D, group also received 1,25(OH)*D, orally in the morning (starting dose 1 pg daily, mean daily dose over the 6 months 0.5 pg)[lO]. Patients in the vitamin D3 group received daily 100 pg vitamin D, orally in the morning. After 6 months of treatment all the patients were studied for another 3 months (t6-t9). At times t_,, to, t3, t6 and tg fasting blood samples were drawn for determination of the serum concentrations of phosphate, alkaline phosphatase and parathyroid hormone (iPTH) using N-terminal and C-terminal specific radioimmunoassays. The serum concentrations of total calcium and creatinine were measured at times t-,, to and thereafter at least thrice weekly during the first 2 weeks, twice monthly for the rest of the treatment period, and at time tg. Bone mineral content
Bone mineral content was measured at times t_,, t,,, t3, t, and tg by photon absorptiometry [‘*‘I], it was calculated as the mean of 12 scans (6 on each forearm) on the distal part of the forearms, and 517
518
CLAUS CHRISTIANSEN
expressed in arbitrary units (g/cm). Forearm bone mineral content values have been shown to correlate well with bone mineral at other locations and with total body calcium [ll]. Bone histomorphometry After double labelling with tetracycline [ 123, transiliac bone biopsy was obtained with an interval of 14 days at time t, and tb. Using the point count principle the following measurements were carried out as described in detail elsewhere [13, 141. Measurements of static parameters in the remodeling of trabecular bone including osteoid surfaces (per cent of total trabecular surfaces), relative osteoid volume (per cent of total trabecular volume) and trabecular osteoclastic resorption surfaces (per cent of total trabecular surfaces). Measurements of dynamic parameters of bone remodeling to evaluate the mineralizing activity including calcification rate in trabecular bone in pm per day as the mean distance between the tetracycline lines in all double labelled zones divided by the number of days between the tetracycline doses, and active trabecular calcification surfaces as the tetracycline surfaces in per cent of total trabecular bone surfaces. Vitamin D measurements Serum concentrations of 250HD, 24,25(OH)2D and l,25(OH)zD were measured according to a previously described method [ 151. This method permits separate determination of the 25-hydroxylated metabolites of vitamin Dz and D3, but in this study we do not report these metabolites separately. The method involves combined measurement of the dihydroxylated metabolites of vitamin D2 and D3, therefore
250HD . 70-
. f
: .
A
:
IO-
Figure 1 gives the individual serum concentrations of the 3 vitamin D metabolites in patients with moderate CRF (n = 15, creatinine clearance 5-35 ml/min), compared to patients with severe CRF
1.25(OH), D
70-
. loo.
T t
:
Initial values
14.0-
7
!
RESULTS
p9lml
6.0-
; 1 . . -A.
Serum and urinary calcium, magnesium, and zinc were measured by atomic absorption spectrophotometry and the serum values were corrected to a constant protein concentration. Serum and urinary creatinine were measured by Technicon SMA 6/60. Serum phosphate and alkaline phosphatase levels were determined by routine laboratory methods. Serum parathyroid hormone (iPTH) was determined by two radioimmunoassays, one being mainly N-terminalspecific and the other mainly C-terminal specific.
“g/ml
. .
30-
Other biochemical measurements
24.25(0H)2 D
n9/ml
50
named 24,25(OH),D and 1,25(OH),D. The method employs specific extraction procedures followed by chromatography on Sephadex LH-20, Lipidex 5000 and high pressure liquid chromatography, internal standards are used to determine recovery of each metabolite. The mean recoveries in the present study were: 24,25(OH),D: 1,25(OH),D: 70% i: 5%; 79% + 4%; and 250HD: 60% _+ 5% (all values given as mean f 1 SD). The metabolites were finally measured by competitive protein binding assays (24,25(OH),D and 1,25(OH),D) or U.V.-detection (250HD). The inter-assay variations estimated by repeated measurements of serum pools in the normal range were 10.5, Il.5 and 9.8%, respectively, and the detection limits were 0.2 ng/ml, 8.3 pg/ml and 4.8 ng/ml.
2.0-
. . . z: Q I
.
Normals o Patients wlh . -
moderate advanced
Fig. 1. Serum levels of vitamin
CRF CRF
D metabolites
in normals
and patients
with chronic
renal failure.
519
Chronic renal failure and vitamin D metabolites SERUM (pg/ml
LO
I
125lOH\O
I I
.
l
30-
l. PC001
20-
.
. .
.*
IO !/ I. I
*
l
IO
P 20 CREA&NE
C&f?ANCE imllmln
)
Fig. 2. Relation between serum 1,25(OH)*D and creatinine clearance in 15 patients with chronic renal failure. (n = 27, creatinine clearance below 5 ml/min), and appropriate controls (n = 24, creatinine clearance above 70 ml/min). The salient features are as follows: no significant difference in 250HD between the 3 groups; normal serum concentration of 24,25(OH)zD in the group with moderate CRF, and an approximately half-normal serum concentration of 24,25(OH)zD in patients with advanced CRF; a strong dependency of renal function on serum 1,25(OH),D, which is further supported by the significant correlation between serum 1,25(OH)*D and creatinine clearance in patients with moderate CRF shown in Fig. 2.
Table 1. Initial BMC and histomorphometric
Tables 1 and 2 show the mean and ranges of the biochemical and histomorphometric values of the patients and controls, and the significance of the differences. The patients demonstrate the characteristic features of moderate renal osteodystrophy as regards both biochemical and histomorphometric values. The histomorphometric measurements showed increased surface area and volume of osteoid in all biopsies and increased surface area of osteoclastic resorption in 6 of 16 biopsies. Linear mineralization was significantly decreased compared to that of controls, whereas the mean area of mineralizing surfaces was normal. The mean values and ranges of the divalent cations in the patients before the start of the trial and in the control groups are shown in Table 3. The mean serum zinc in the patients was significantly lower than in the controls (P < 0.001). This hypozincemia was caused by a reduction in the albumin bound fraction, while the cc,-macroglobulin fraction was virtually normal [16]. Furthermore, the patients as a group had hypocalcemia (P < 0.01) and hypermagnesemia (P < 0.01). The urinary excretion rates of zinc and magnesium were slightly, but not significantly, increased, while the urinary excretion of calcium was significantly decreased. Spontaneous course The initial values and the spontaneous course in the first 6 months of the study are detailed in Table 4. Except for small changes in iPTH, the mild renal
values in patients with moderate chronic renal failure compared to normal values (sex and age matched) Patients
Bone mineral content (% of normal) Osteoid surfaces (%) Osteoid volume (%) Bone resorption (%) Calcification rate (pm/d) Active trabecular calcification surfaces (%)
Normals
n
mean
range
n
mean
range
Significance of difference
17
75.3
(53-86)
146
100
-
P < 0.001
16 16 16 16 16
74 13.2 9.6 0.35 15.7
(51-93) (6.622.6) (4-31) (cbO.7) (l-45)
17 17 17 8 8
20 1.5 4.7 0.62 16.9
(7-30) (0.2-3.7) (2-8) (0.54.7) (S-27)
P P P P
< < < <
0.001 0.001 0.01 0.01
NS
Table 2. Initial serum levels of substances important in mineral metabolism in patients with moderate chronic renal failure compared to normal values Patients
S-phosphate (mmol/l) S-alkaline phosphatase (U/l) S-iPTH (N-terminal) (pg/l) S-iPTH (C-terminal) (pg/l) S-L25(OH)2D (pg/ml) S-24,25(OH),D (ng/ml) S-250HD (ng/ml)
Normals
n
mean
range
n
mean
range
17 17 17 17 15 15 15
1.4 221 0.23 2.4 17.7 2.83 23.2
(l&2.4) (114-452) (0.03-l .23) (0.55-6.9) (c-34.5) (0.2-7. I) (8.4-48.6)
99 194 28 102 24 24 24
1.1 140 0.04 0.34 28.6 2.84 26.1
(0.8-1.4) < 220 < 0.08 co.55 (13.2-47.6) (0.734.9) (1 l&47.5)
Significance of difference P P P P P
< < < < <
0.001 0.001
0.001 0.001 0.05 NS NS
CLAUS CHRISTIANSEN
520
Table 3. Initial concentrations of divalent cations in patients with moderate chronic renal failure compared with values in normal subjects Patients
S-calcium (mmol/l) S-magnesium (mmol/l) S-zinc (pmol/l) U-calcium (mmol/mmol creatinine) U-magnesium (mmol/mmol creatinine) U-zinc (~mol/mmol creatinine)
Normals
n
mean
range
n
mean
range
Significance of difference
17 17 17
2.34 1.04 12.9
(2.01-2.69) (0.45-1.62) (11.3-14.6)
194 194 104
2.47 0.83 14.5
(2.33-2.60) (0.734.94) (10.4-18.4)
P < 0.01 P < 0.01 P
17
0.15
(0.04-0.39)
28
0.43
(0.1 lkO.75)
P < 0.001
17 17
0.48 0.73
(0.02-0.99) (0.03-1.55)
28 28
0.37 0.52
(0.1(M.63) (0.04-l .06)
NS NS
osteodystrophy and the renal function were virtually unchanged over the observation period. Effect of treatment The effects of treatment with 1,25(OH)aD, or Ds on the measured variables are given in Tables 5, 6 and 7. Mineral metabolism 1,25(OH)sD, caused a reduction the resorption surfaces in trabecular
of osteoid
and of
bone, but had no effect on forearm bone mineral content. On the other hand, vitamin D had no effects on bone morphometry or bone mineral content (Table 5).
In the 1,25(OH),D, group the mean serum concentration of 1,25(OH)zD rose significantly during treatment, whereas serum concentrations of 250HD and 24,25(OH),D remained unchanged. In the D, group there was a highly significant increase in serum concentrations of 250HD and 24,25(OH)*D, whereas 1,25(OH)2D remained unchanged. The changes in the two groups of mean serum levels of iPTH and alkaline phosphatase were similar but most marked in the 1,25(OH)zD3 group (Table 6). The serum concentrations of the divalent cations expressed as a function of treatment are shown in Table 7. A significant increase in serum calcium was found in the group treated with 1,2.5(OH)2D, in con-
Table 4. Spontaneous course for 6 months in patients with moderate chronic renal failure
to
(mean f SD)
(mean + SD)
Significance of difference
75.3 + 9.4 20.8 + 14.8 2.07 + 1.83 19.8 + 19.6 0.20 _+0.29 1.63 + 1.55 60.2 & 47.7 22.7 f 10.9
74.6 k 11.2 23.2 k 12.0 2.83 + 2.17 17.7 k 11.6 0.29 It: 0.36 2.60 + 2.16 54.1 f 42.8 22.4 k 11.9
NS NS NS NS P < 0.05 P < 0.01 NS NS
t-6
BMC (% of normal) 250HD (ng/ml) 24,25(OH)2D (ng/ml) l,25(OH)2D (pg/ml) iPTH (N-terminal) (pg/l) iPTH (C-terminal) @g/l) 24 h Calcium/cr. (mg/g) Creatinine clear. (ml/min)
Table 5. Effect of treatment for 6 months in 2 groups of patients with moderate chronic renal failure 1,25(OH),D, group
Bone mineral content (% of normal) Osteoid surfaces (%) Osteoid volume (%) Bone resorption (%) Calcification rate (pm/d) Active trabecular calcification surfaces (%) * = P < 0.05. ** = P < 0.01
Vitamin D3 group
ro (mean f SD)
(meant6& SD)
(mean k SD)
to
L5 (mean &SD)
81.3 k 5.1
80.5 f 5.7
70.2 & 12.3
68.0 + 10.6
77.1 f 8.8 12.3 _+4.8 10.7 * 9.0 0.41 & 0.15 17.4 * 13.3
75.5 * 14.1 Il.3 & 4.8 7.7 & 5.9 0.43 f 0.20 26.6 k 23.9
67.4 k 15.4 13.4 f 4.5 8.6 + 4.4 0.30 & 0.17 14.0 & 10.6
63.1 + 7.4** + 4.08 + 0.33 + 19.4 f
16.8 2.0 1.4 0.20 26.2
Chronic renal failure and vitamin D metabolites
521
Table 6. Mean changes (per cent of initial values) in serum substances important metabolism in patients with moderate chronic renal failure
vitamin D3 group
l,25(OH),Da group
+ treatment
+ treatment r6
r3 +3 -2811 -421 -4011 + 54r -5 -24
Phosphate Alkaline phosphatase iPTH (N-terminal) iPTH (C-terminal) 1,25(OH)*D 24,25(OH)zD 250HD
+6
-2611 -431 -5411 + 637 +4 -34
in mineral
-treatment r9
r3
-6 -16 -20 -31 -3 -3
f6 -1 -19 -211 f20 +97t + 18211
-treatment r9
r,
f8 -15 -331 -29 + 671 + 14511
f3 -13 - 391 -2SJl f6 flllTT +227fT
1 = P < 0.05. 11 = P < 0.01.
Table 7. Mean changes (per cent of initial values) in divalent cations important
in mineral
metabolism in patients with moderate chronic renal failure Vitamin Ds group + treatment -treatment r3 t9 rs
l,25(OH)2Ds group + treatment -treatment rs r9 r3 Serum Urinary Excretion Rate
Calcium Magnesium Zinc Calcium Magnesium Zinc
+8 - 5 +3 + 134rt +15 + 34t
+ 12?f -1 -4 +310f +8 +31
-1 -2 0 +118 -12 +26
0 +2 +4 +5 -4 f20
0 + 10 +gT +4 -13 0
fl +13 +3 + 54T +11 +21
1 = P < 0.05. I]: = P < 0.01.
trast to the group treated with D,. After withdrawal the serum calcium level returned to the initial value. In both treatment groups the serum zinc and magnesium were unchanged during the study. There was a 300% increase in urinary calcium excretion rate in the group treated with 1,25(OH),D,. The urinary calcium excretion rate in this group returned to the initial value after withdrawal. A similar, but much less pronounced pattern was observed in the D3 group. In both treatment groups there was a tendency towards an increased urinary excretion rate of zinc during the treatment period. The urinary excretion rate of magnesium was constant in both groups during treatment.
L.?S(OH)~-vifomin
100
O:, group
---___
90
3U 80 S 5 70
1 p401
7
9
‘;6Oq /
,
,
>
I
3
\: .
Renal function The mean creatinine clearance was similar in the 2 subgroups and virtually unchanged in the 6 months preceding therapy. During 6 months treatment there was a reduction of creatinine clearance in both groups. Renal function deteriorated more during treatment with l,25(OH)zD, than during the preceding 6 months (P < 0.01) (Fig. 3).
b
I2 Monihs
b
kJ
tUnrreotedtTreofed--1 to
DISCUSSION We examined the therapeutic and Ds in undialysed patients
role of 1,25(OH)sD, with CRF in a con-
Fig. 3. Mean percentage reduction in creatinine clearance before and during treatment with 1,25(OH)*D, and vitamin D,.
522
CLAUS CHRISTIANSEN
trolled study. The patients studied had a creatinine clearance of less than 35 ml/min and biochemical evidence of mild renal osteodystrophy. Previously published reports suggest that such patients have bone histomorphometric evidence of renal osteodystrophy 18,171. Others [S-S] have also found that treatment with even small doses of 1,25(OH)2D, (or for that matter lc(-OHD,) caused a considerable increase in the serum concentration and urinary excretion of calcium, and a fall in serum iPTH and alkaline phosphatase. In 7 out of 8 patients in the 1,25(0H),D-group concentrations of N-terminal iPTH reached the normal range. With the dose of vitamin D3 given there was a slight increase in serum calcium and urinary calcium excretion and a moderate fall in serum iPTH. The present study confirms that treatment with 1,25(OH)2D3 (and to a smaller degree D, also) will reduce the abnormalities of blood mineral metabolism and bone remodeling, found in chronic renal failure. Although the skeletal evidence of renal osteodystrophy was not very pronounced in our patients, 1,25(OH)2D3 did not bring the histomorphometry to normal values in several patients. The treatment failed to increase the bone mineral content in the forearms. Our results demonstrate that treatment of patients in CRF with 1,25(OH)2D3 in the dose given does not influence serum concentrations of 250HD and 24,25(OH),D. We know from animal experiments that the administration of 1,25(OH),D, stimulates the production of 24,25(OH)2D, but these results were obtained in vitamin D deficient rats without uremia [IS]. The changes of serum creatinine seen in our patients reflect changes in renal function, as discussed elsewhere [lo]. Our data demonstrate that spontaneous changes cannot explain these findings, since mean serum creatinine rose sharply at the beginning, and fell at the end, of treatment [19]. It is well-known that hypercalcemia implies a deterioration in renal function. When uremic undialysed patients are treated with the newer vitamin D anaiogues the aim is to cure the renal bone disease and concomitant hyperparathyroidism frequently found in such patients. Actually, the fall in iPTH is used as an indicator of successful treatment [20,21]. When 1,25(OH)*D3 is given to uremic patients in more than homeopathic doses the intestinal absorption of calcium increases, and so does the renal load of calcium. Our data show that even small doses of 1,25(OH)2D, in such patients lead to an inevitable rise in serum calcium, although the patients cannot be regarded as having vitamin D intoxication as traditionally defined by serum calcium levels. These minimal increases in serum calcium are closely related to the therapeutic effect on calcium metabolism as judged by the fall in iPTH. Furthermore our data show that the minor changes in serum calcium seen in the 1,25(OH),D,-group are correlated to increased values of serum creatinine.
Stanbury has warned against the indiscriminate administration of D3 (or its new analogues) to patients with CRF [4]. However, DeLuca has pointed out the possibility that administration of 1,25(OH)zD, or its analogues may prevent the onset of this bone disease if administered early in the course of renal failure. Our present results lend strong support to Stanbury’s opinion and suggest another possibility, that biochemical changes in calcium and phosphorus metabolism seen in CRF are beneficial to the patients in protecting them against extraskeletal calcification. Correction of the disturbances of mineral metabolism in CRF may put renal function at risk. Consequently, the decreased formation of 1,25(OH)zD3 in renal failure can be regarded as an appropriate and important defence mechanism in CRF. Treatment with la-hydroxylated vitamin D analogues should be restricted to the relatively few patients with severe symptomatic renal osteodystrophy. Acknowledgements--I am indebted to Dr M. Uskokovic of Hoffmann-La Roche, Nutley, for supply of 1,25(OH),D, and 24,2S(OH),D3 for assay purpose; to Dr J. Guncaga of Hoffmann-La Roche, Basel, for supply of 1,25(OH)*D, and vitamin D, employed in this study; to Dr F. Melsen for performing the bone histomorphy; and to Dr M. S. Christensen for performing the iPTH (N-terminal) analyses. The study was supported by grants from the Danish Medical Research Council (j.nos. 512-9091, 512-10210).
REFERENCES
1. Fraser D. R. and Kodicek E.: Unique biosynthesis by kidney of a biologically active vitamin D metabolite. Nature 228 (1970) 764766. 2. Mawer E. B., Backhouse J., Taylor C. M., Lumb G. A. and Stanbury S. W.: Failure of formation of 1,25-dihydroxycholecalciferol in chronic renal insufficiency. Lancet i (1973) 626-628. DeL&a H. F.: Vitamin D metabolism. C/in. Endocr. 7 suppl. (1977) ls-17s. Stanbury S. W.: The role of vitamin D in renal bone disease. C/in. Endocr. 7 suppl. (1977) 25s30s. Brickman A. S., Coburn J. W. and Norman A. W.: Action of 1,2S-dihydroxycholecalciferol, a potent kidney-produced metabolite of vitamin D3 in uremic man. New Engl. J. Med. 287 (1972) 891-895. R. G., Russell R. G. G., Ledingham 6. Hendersen J. G. G., Smith R., Oliver D. O., Walton R. J., Small D. G., Preston C., Warner G. T. and Norman A. W.: Effects of l,25-dihydroxycholecalciferol on calcium absorption, muscle weakness, and bone disease in chronic renal failure. Lancet i (I 974) 379-384. I. Pierides A. M., Ellis H. A., Simpson W., Cook D. and Kerr D. N. S.: The effect of lee-hydroxyvitamin D3 in predialysis renal bone disease. C/in. Endocr. 7 suppl (1977) 109~116s. M. S.: Bone 8. Melsen F., Nielsen H. E. and Christensen histomorphometry in patients with chronic renal failure: effect of la-hydroxyvitamin D,. C/in. Endocr. 7 suppl (1977) 39+44s. 9. Tougaard L., Sorensen E., Brschner-Mortensen J., Christensen M. S., Rsdbro P. and Sorensen A. W. S.: Controlled trial of la-hydroxycholecalciferol in chronic renal failure. Lancer 1 (1976) 1044-1047.
Chronic renal failure and vitamin D metabolites 10. Christiansen C., Rsdbro P., Christensen M. S., Hartnack B. and Transbel I.: Deterioration of renal function during treatment of chronic renal failure with 1,25-dihydroxycholecalciferol. Lancet ii (1978) 700703. 11. Christiansen C., Redbro P. and Jensen H.: Bone minera1 content in the forearm measured by photon absorptiometry: principles and reliability. Stand. J. c/in. Lab. Invest. 35 (1975) 323-330. 12. Melsen F., Melsen B. and Mosekilde L.: An evaluation of the quantitative parameters applied in bone histology. Acta path. microbial. stand. Sect. A 86 (1978) 63-69. 13. Melsen F., Melsen B., Mosekilde L. and Bergman S.: Histomorphometric analysis of normal bone from the iliac creast. Acta path. microbial. stand. Sect. A 86 (1978) 7&81. 14. Melsen F. and Mosekilde L.: Tetracycline double labelling of iliac bone in 41 normal adults. Calcif: Tim Res. 26 (1978) 99-102. 15. Shepard P. M., Horst R. L., Jorgensen N. A. and DeLuca H. F.: Determination of vitamin D and its metabolites in plasma from normal and anephric man. Biochem. J. 182 (1979) 55-69.
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16. Kiilerich S., Christiansen C., Christensen M. S. and Nzstoft J.: Zinc metabolism in patients with chronic renal failure during treatment with 1,25-dihydroxycholecalciferol: a controlled therapeutic trial. Clin. Nephrol. 15 (1981) 23-27. 17. Malluche H. H., Ritz E., Lange H. P., Kutschera J., Hodgson M., SeitTert V. and Schoppe W.: Bone histology in incipient and advanced renal failure. Kidney Int. 9 (1976) 355-360. 18. Tanaka Y. and DeLuca H. F.: Stimulation of 24,25_dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3. Science 183 (1974) 1198-1200. 19. Christiansen C., Rerdbro P., Christensen M. S. and Hartnack B.: Is 1,25-dihydroxy-cholecalciferol harmful to renal function in patients with chronic renal failure? Clin. Endocr. 15 (1981) 229-236. 20. Fischer J. A. and Binswamzer U.: 1.2%dihvdroxvchole. < calciferol in dialysed patients with clinically asymptomatic renal osteodystrophy. A controlled study. Cow. Nephrol. 18 (1980) 82-91. 21. Malluche H. H., Goldstein D. A. and Massry S. G.: Effects of 6 months therapy with 1,25(OH),D, on bone disease of dialysis patients. Contr. Nephrol. 18 (1980) 9&104.
1983 Copyright
0022-473 l/83 $3.00 + 0.00 0 1983 Pergamon Press Ltd
CHRONIC RENAL FAILURE AND VITAMIN D METABOLITES: A STATUS REPORT CLAUSCHR~~TIANSEN Department of Clinical Chemistry, Glostrup Hospital, University of Copenhagen, Glostrup, Denmark
SUMMARY Seventeen undialysed adult patients with chronic renal failure took part in a controlled study of the effects of 1,25(OH),D, and D3. After a 6-month observation period the patients were allocated at random to 2 groups for 6 months of treatment with either 1,25(OH),D, (mean dose 0.5 pg daily) or D3 (dose 1OOpgdaily). Treatment was then discontinued and the patients were studied for a further 3 months. The patients as a group initially had a moderate renal osteodystrophy. During treatment serum iPTH decreased in both groups but most markedly in the 1,25(OH)2Dogroup in which the iPTH values became normal. Serum creatinine increased during treatment in both groups, in the group receiving 1,25(OH),D, this was coupled with an increase in serum calcium to within the normal range. Our data demonstrate that 1,25(OH),Ds treatment in patients with chronic renal failure leads to a further reduction in renal function, which may be partially reversible. Physicians should therefore be reluctant to give vitamin D analogues to patients with chronic renal failure unless they have severe symptomatic renal osteodystrophy.
METHODS
INTRODUCTION Decreased or absent la-hydroxylating activity is seen in progressive chronic renal failure (CRF) [l, 21, and is believed to be important in the development of renal osteodystrophy [3,4]. A number of uncontrolled studies have been published concerning the effects of the vitamin D analogues la-hydroxycholecalciferol (la-OHD,) and 1,25-dihydroxycholecalciferal (1,25(OH),D,) on various aspects of calcium and phosphorus metabolism, including a number of observations on the effect on bone histology [SS]. Most of the reports are positive, some even enthusiastic. Hypercalcemia is a well-known complication to therapy, and deterioration of renal function in CRF has been suggested in a controlled study of la-0HD3 in predialysis patients [9]. We have compared the effects of 1,25(OH)*D3 and vitamin D, in a controlled study of predialysis CRF patients with special reference to a possible effect on both bone and biochemical parameters. as well as renal function.
PATIENTS The study comprised of 17 patients with chronic renal failure. They were 5 men and 12 women aged 26-70 yr (mean age 53 yr). Renal function had been relatively stable for more than 1 yr and the creatinine clearance. was between 5 and 35 ml/min. Details of the selection criteria have been given elsewhere [lo].
Design of study
After the preliminary investigations (t-a) the patients were studied for 6 months (until to) to determine the spontaneous course in the observed parameters. The 17 patients were randomly allocated into two groups: one was treated with 1,25(OH)*D3, and the other with vitamin D, (see below). During the following 6 months (to-Q all patients received 0.5 g calcium daily. Patients in the 1,25(OH)*D, group also received 1,25(OH)*D, orally in the morning (starting dose 1 pg daily, mean daily dose over the 6 months 0.5 pg)[lO]. Patients in the vitamin D3 group received daily 100 pg vitamin D, orally in the morning. After 6 months of treatment all the patients were studied for another 3 months (t6-t9). At times t_,, to, t3, t6 and tg fasting blood samples were drawn for determination of the serum concentrations of phosphate, alkaline phosphatase and parathyroid hormone (iPTH) using N-terminal and C-terminal specific radioimmunoassays. The serum concentrations of total calcium and creatinine were measured at times t-,, to and thereafter at least thrice weekly during the first 2 weeks, twice monthly for the rest of the treatment period, and at time tg. Bone mineral content
Bone mineral content was measured at times t_,, t,,, t3, t, and tg by photon absorptiometry [‘*‘I], it was calculated as the mean of 12 scans (6 on each forearm) on the distal part of the forearms, and 517
518
CLAUS CHRISTIANSEN
expressed in arbitrary units (g/cm). Forearm bone mineral content values have been shown to correlate well with bone mineral at other locations and with total body calcium [ll]. Bone histomorphometry After double labelling with tetracycline [ 123, transiliac bone biopsy was obtained with an interval of 14 days at time t, and tb. Using the point count principle the following measurements were carried out as described in detail elsewhere [13, 141. Measurements of static parameters in the remodeling of trabecular bone including osteoid surfaces (per cent of total trabecular surfaces), relative osteoid volume (per cent of total trabecular volume) and trabecular osteoclastic resorption surfaces (per cent of total trabecular surfaces). Measurements of dynamic parameters of bone remodeling to evaluate the mineralizing activity including calcification rate in trabecular bone in pm per day as the mean distance between the tetracycline lines in all double labelled zones divided by the number of days between the tetracycline doses, and active trabecular calcification surfaces as the tetracycline surfaces in per cent of total trabecular bone surfaces. Vitamin D measurements Serum concentrations of 250HD, 24,25(OH)2D and l,25(OH)zD were measured according to a previously described method [ 151. This method permits separate determination of the 25-hydroxylated metabolites of vitamin Dz and D3, but in this study we do not report these metabolites separately. The method involves combined measurement of the dihydroxylated metabolites of vitamin D2 and D3, therefore
250HD . 70-
. f
: .
A
:
IO-
Figure 1 gives the individual serum concentrations of the 3 vitamin D metabolites in patients with moderate CRF (n = 15, creatinine clearance 5-35 ml/min), compared to patients with severe CRF
1.25(OH), D
70-
. loo.
T t
:
Initial values
14.0-
7
!
RESULTS
p9lml
6.0-
; 1 . . -A.
Serum and urinary calcium, magnesium, and zinc were measured by atomic absorption spectrophotometry and the serum values were corrected to a constant protein concentration. Serum and urinary creatinine were measured by Technicon SMA 6/60. Serum phosphate and alkaline phosphatase levels were determined by routine laboratory methods. Serum parathyroid hormone (iPTH) was determined by two radioimmunoassays, one being mainly N-terminalspecific and the other mainly C-terminal specific.
“g/ml
. .
30-
Other biochemical measurements
24.25(0H)2 D
n9/ml
50
named 24,25(OH),D and 1,25(OH),D. The method employs specific extraction procedures followed by chromatography on Sephadex LH-20, Lipidex 5000 and high pressure liquid chromatography, internal standards are used to determine recovery of each metabolite. The mean recoveries in the present study were: 24,25(OH),D: 1,25(OH),D: 70% i: 5%; 79% + 4%; and 250HD: 60% _+ 5% (all values given as mean f 1 SD). The metabolites were finally measured by competitive protein binding assays (24,25(OH),D and 1,25(OH),D) or U.V.-detection (250HD). The inter-assay variations estimated by repeated measurements of serum pools in the normal range were 10.5, Il.5 and 9.8%, respectively, and the detection limits were 0.2 ng/ml, 8.3 pg/ml and 4.8 ng/ml.
2.0-
. . . z: Q I
.
Normals o Patients wlh . -
moderate advanced
Fig. 1. Serum levels of vitamin
CRF CRF
D metabolites
in normals
and patients
with chronic
renal failure.
519
Chronic renal failure and vitamin D metabolites SERUM (pg/ml
LO
I
125lOH\O
I I
.
l
30-
l. PC001
20-
.
. .
.*
IO !/ I. I
*
l
IO
P 20 CREA&NE
C&f?ANCE imllmln
)
Fig. 2. Relation between serum 1,25(OH)*D and creatinine clearance in 15 patients with chronic renal failure. (n = 27, creatinine clearance below 5 ml/min), and appropriate controls (n = 24, creatinine clearance above 70 ml/min). The salient features are as follows: no significant difference in 250HD between the 3 groups; normal serum concentration of 24,25(OH)zD in the group with moderate CRF, and an approximately half-normal serum concentration of 24,25(OH)zD in patients with advanced CRF; a strong dependency of renal function on serum 1,25(OH),D, which is further supported by the significant correlation between serum 1,25(OH)*D and creatinine clearance in patients with moderate CRF shown in Fig. 2.
Table 1. Initial BMC and histomorphometric
Tables 1 and 2 show the mean and ranges of the biochemical and histomorphometric values of the patients and controls, and the significance of the differences. The patients demonstrate the characteristic features of moderate renal osteodystrophy as regards both biochemical and histomorphometric values. The histomorphometric measurements showed increased surface area and volume of osteoid in all biopsies and increased surface area of osteoclastic resorption in 6 of 16 biopsies. Linear mineralization was significantly decreased compared to that of controls, whereas the mean area of mineralizing surfaces was normal. The mean values and ranges of the divalent cations in the patients before the start of the trial and in the control groups are shown in Table 3. The mean serum zinc in the patients was significantly lower than in the controls (P < 0.001). This hypozincemia was caused by a reduction in the albumin bound fraction, while the cc,-macroglobulin fraction was virtually normal [16]. Furthermore, the patients as a group had hypocalcemia (P < 0.01) and hypermagnesemia (P < 0.01). The urinary excretion rates of zinc and magnesium were slightly, but not significantly, increased, while the urinary excretion of calcium was significantly decreased. Spontaneous course The initial values and the spontaneous course in the first 6 months of the study are detailed in Table 4. Except for small changes in iPTH, the mild renal
values in patients with moderate chronic renal failure compared to normal values (sex and age matched) Patients
Bone mineral content (% of normal) Osteoid surfaces (%) Osteoid volume (%) Bone resorption (%) Calcification rate (pm/d) Active trabecular calcification surfaces (%)
Normals
n
mean
range
n
mean
range
Significance of difference
17
75.3
(53-86)
146
100
-
P < 0.001
16 16 16 16 16
74 13.2 9.6 0.35 15.7
(51-93) (6.622.6) (4-31) (cbO.7) (l-45)
17 17 17 8 8
20 1.5 4.7 0.62 16.9
(7-30) (0.2-3.7) (2-8) (0.54.7) (S-27)
P P P P
< < < <
0.001 0.001 0.01 0.01
NS
Table 2. Initial serum levels of substances important in mineral metabolism in patients with moderate chronic renal failure compared to normal values Patients
S-phosphate (mmol/l) S-alkaline phosphatase (U/l) S-iPTH (N-terminal) (pg/l) S-iPTH (C-terminal) (pg/l) S-L25(OH)2D (pg/ml) S-24,25(OH),D (ng/ml) S-250HD (ng/ml)
Normals
n
mean
range
n
mean
range
17 17 17 17 15 15 15
1.4 221 0.23 2.4 17.7 2.83 23.2
(l&2.4) (114-452) (0.03-l .23) (0.55-6.9) (c-34.5) (0.2-7. I) (8.4-48.6)
99 194 28 102 24 24 24
1.1 140 0.04 0.34 28.6 2.84 26.1
(0.8-1.4) < 220 < 0.08 co.55 (13.2-47.6) (0.734.9) (1 l&47.5)
Significance of difference P P P P P
< < < < <
0.001 0.001
0.001 0.001 0.05 NS NS
CLAUS CHRISTIANSEN
520
Table 3. Initial concentrations of divalent cations in patients with moderate chronic renal failure compared with values in normal subjects Patients
S-calcium (mmol/l) S-magnesium (mmol/l) S-zinc (pmol/l) U-calcium (mmol/mmol creatinine) U-magnesium (mmol/mmol creatinine) U-zinc (~mol/mmol creatinine)
Normals
n
mean
range
n
mean
range
Significance of difference
17 17 17
2.34 1.04 12.9
(2.01-2.69) (0.45-1.62) (11.3-14.6)
194 194 104
2.47 0.83 14.5
(2.33-2.60) (0.734.94) (10.4-18.4)
P < 0.01 P < 0.01 P
17
0.15
(0.04-0.39)
28
0.43
(0.1 lkO.75)
P < 0.001
17 17
0.48 0.73
(0.02-0.99) (0.03-1.55)
28 28
0.37 0.52
(0.1(M.63) (0.04-l .06)
NS NS
osteodystrophy and the renal function were virtually unchanged over the observation period. Effect of treatment The effects of treatment with 1,25(OH)aD, or Ds on the measured variables are given in Tables 5, 6 and 7. Mineral metabolism 1,25(OH)sD, caused a reduction the resorption surfaces in trabecular
of osteoid
and of
bone, but had no effect on forearm bone mineral content. On the other hand, vitamin D had no effects on bone morphometry or bone mineral content (Table 5).
In the 1,25(OH),D, group the mean serum concentration of 1,25(OH)zD rose significantly during treatment, whereas serum concentrations of 250HD and 24,25(OH),D remained unchanged. In the D, group there was a highly significant increase in serum concentrations of 250HD and 24,25(OH)*D, whereas 1,25(OH)2D remained unchanged. The changes in the two groups of mean serum levels of iPTH and alkaline phosphatase were similar but most marked in the 1,25(OH)zD3 group (Table 6). The serum concentrations of the divalent cations expressed as a function of treatment are shown in Table 7. A significant increase in serum calcium was found in the group treated with 1,2.5(OH)2D, in con-
Table 4. Spontaneous course for 6 months in patients with moderate chronic renal failure
to
(mean f SD)
(mean + SD)
Significance of difference
75.3 + 9.4 20.8 + 14.8 2.07 + 1.83 19.8 + 19.6 0.20 _+0.29 1.63 + 1.55 60.2 & 47.7 22.7 f 10.9
74.6 k 11.2 23.2 k 12.0 2.83 + 2.17 17.7 k 11.6 0.29 It: 0.36 2.60 + 2.16 54.1 f 42.8 22.4 k 11.9
NS NS NS NS P < 0.05 P < 0.01 NS NS
t-6
BMC (% of normal) 250HD (ng/ml) 24,25(OH)2D (ng/ml) l,25(OH)2D (pg/ml) iPTH (N-terminal) (pg/l) iPTH (C-terminal) @g/l) 24 h Calcium/cr. (mg/g) Creatinine clear. (ml/min)
Table 5. Effect of treatment for 6 months in 2 groups of patients with moderate chronic renal failure 1,25(OH),D, group
Bone mineral content (% of normal) Osteoid surfaces (%) Osteoid volume (%) Bone resorption (%) Calcification rate (pm/d) Active trabecular calcification surfaces (%) * = P < 0.05. ** = P < 0.01
Vitamin D3 group
ro (mean f SD)
(meant6& SD)
(mean k SD)
to
L5 (mean &SD)
81.3 k 5.1
80.5 f 5.7
70.2 & 12.3
68.0 + 10.6
77.1 f 8.8 12.3 _+4.8 10.7 * 9.0 0.41 & 0.15 17.4 * 13.3
75.5 * 14.1 Il.3 & 4.8 7.7 & 5.9 0.43 f 0.20 26.6 k 23.9
67.4 k 15.4 13.4 f 4.5 8.6 + 4.4 0.30 & 0.17 14.0 & 10.6
63.1 + 7.4** + 4.08 + 0.33 + 19.4 f
16.8 2.0 1.4 0.20 26.2
Chronic renal failure and vitamin D metabolites
521
Table 6. Mean changes (per cent of initial values) in serum substances important metabolism in patients with moderate chronic renal failure
vitamin D3 group
l,25(OH),Da group
+ treatment
+ treatment r6
r3 +3 -2811 -421 -4011 + 54r -5 -24
Phosphate Alkaline phosphatase iPTH (N-terminal) iPTH (C-terminal) 1,25(OH)*D 24,25(OH)zD 250HD
+6
-2611 -431 -5411 + 637 +4 -34
in mineral
-treatment r9
r3
-6 -16 -20 -31 -3 -3
f6 -1 -19 -211 f20 +97t + 18211
-treatment r9
r,
f8 -15 -331 -29 + 671 + 14511
f3 -13 - 391 -2SJl f6 flllTT +227fT
1 = P < 0.05. 11 = P < 0.01.
Table 7. Mean changes (per cent of initial values) in divalent cations important
in mineral
metabolism in patients with moderate chronic renal failure Vitamin Ds group + treatment -treatment r3 t9 rs
l,25(OH)2Ds group + treatment -treatment rs r9 r3 Serum Urinary Excretion Rate
Calcium Magnesium Zinc Calcium Magnesium Zinc
+8 - 5 +3 + 134rt +15 + 34t
+ 12?f -1 -4 +310f +8 +31
-1 -2 0 +118 -12 +26
0 +2 +4 +5 -4 f20
0 + 10 +gT +4 -13 0
fl +13 +3 + 54T +11 +21
1 = P < 0.05. I]: = P < 0.01.
trast to the group treated with D,. After withdrawal the serum calcium level returned to the initial value. In both treatment groups the serum zinc and magnesium were unchanged during the study. There was a 300% increase in urinary calcium excretion rate in the group treated with 1,25(OH),D,. The urinary calcium excretion rate in this group returned to the initial value after withdrawal. A similar, but much less pronounced pattern was observed in the D3 group. In both treatment groups there was a tendency towards an increased urinary excretion rate of zinc during the treatment period. The urinary excretion rate of magnesium was constant in both groups during treatment.
L.?S(OH)~-vifomin
100
O:, group
---___
90
3U 80 S 5 70
1 p401
7
9
‘;6Oq /
,
,
>
I
3
\: .
Renal function The mean creatinine clearance was similar in the 2 subgroups and virtually unchanged in the 6 months preceding therapy. During 6 months treatment there was a reduction of creatinine clearance in both groups. Renal function deteriorated more during treatment with l,25(OH)zD, than during the preceding 6 months (P < 0.01) (Fig. 3).
b
I2 Monihs
b
kJ
tUnrreotedtTreofed--1 to
DISCUSSION We examined the therapeutic and Ds in undialysed patients
role of 1,25(OH)sD, with CRF in a con-
Fig. 3. Mean percentage reduction in creatinine clearance before and during treatment with 1,25(OH)*D, and vitamin D,.
522
CLAUS CHRISTIANSEN
trolled study. The patients studied had a creatinine clearance of less than 35 ml/min and biochemical evidence of mild renal osteodystrophy. Previously published reports suggest that such patients have bone histomorphometric evidence of renal osteodystrophy 18,171. Others [S-S] have also found that treatment with even small doses of 1,25(OH)2D, (or for that matter lc(-OHD,) caused a considerable increase in the serum concentration and urinary excretion of calcium, and a fall in serum iPTH and alkaline phosphatase. In 7 out of 8 patients in the 1,25(0H),D-group concentrations of N-terminal iPTH reached the normal range. With the dose of vitamin D3 given there was a slight increase in serum calcium and urinary calcium excretion and a moderate fall in serum iPTH. The present study confirms that treatment with 1,25(OH)2D3 (and to a smaller degree D, also) will reduce the abnormalities of blood mineral metabolism and bone remodeling, found in chronic renal failure. Although the skeletal evidence of renal osteodystrophy was not very pronounced in our patients, 1,25(OH)2D3 did not bring the histomorphometry to normal values in several patients. The treatment failed to increase the bone mineral content in the forearms. Our results demonstrate that treatment of patients in CRF with 1,25(OH)2D3 in the dose given does not influence serum concentrations of 250HD and 24,25(OH),D. We know from animal experiments that the administration of 1,25(OH),D, stimulates the production of 24,25(OH)2D, but these results were obtained in vitamin D deficient rats without uremia [IS]. The changes of serum creatinine seen in our patients reflect changes in renal function, as discussed elsewhere [lo]. Our data demonstrate that spontaneous changes cannot explain these findings, since mean serum creatinine rose sharply at the beginning, and fell at the end, of treatment [19]. It is well-known that hypercalcemia implies a deterioration in renal function. When uremic undialysed patients are treated with the newer vitamin D anaiogues the aim is to cure the renal bone disease and concomitant hyperparathyroidism frequently found in such patients. Actually, the fall in iPTH is used as an indicator of successful treatment [20,21]. When 1,25(OH)*D3 is given to uremic patients in more than homeopathic doses the intestinal absorption of calcium increases, and so does the renal load of calcium. Our data show that even small doses of 1,25(OH)2D, in such patients lead to an inevitable rise in serum calcium, although the patients cannot be regarded as having vitamin D intoxication as traditionally defined by serum calcium levels. These minimal increases in serum calcium are closely related to the therapeutic effect on calcium metabolism as judged by the fall in iPTH. Furthermore our data show that the minor changes in serum calcium seen in the 1,25(OH),D,-group are correlated to increased values of serum creatinine.
Stanbury has warned against the indiscriminate administration of D3 (or its new analogues) to patients with CRF [4]. However, DeLuca has pointed out the possibility that administration of 1,25(OH)zD, or its analogues may prevent the onset of this bone disease if administered early in the course of renal failure. Our present results lend strong support to Stanbury’s opinion and suggest another possibility, that biochemical changes in calcium and phosphorus metabolism seen in CRF are beneficial to the patients in protecting them against extraskeletal calcification. Correction of the disturbances of mineral metabolism in CRF may put renal function at risk. Consequently, the decreased formation of 1,25(OH)zD3 in renal failure can be regarded as an appropriate and important defence mechanism in CRF. Treatment with la-hydroxylated vitamin D analogues should be restricted to the relatively few patients with severe symptomatic renal osteodystrophy. Acknowledgements--I am indebted to Dr M. Uskokovic of Hoffmann-La Roche, Nutley, for supply of 1,25(OH),D, and 24,2S(OH),D3 for assay purpose; to Dr J. Guncaga of Hoffmann-La Roche, Basel, for supply of 1,25(OH)*D, and vitamin D, employed in this study; to Dr F. Melsen for performing the bone histomorphy; and to Dr M. S. Christensen for performing the iPTH (N-terminal) analyses. The study was supported by grants from the Danish Medical Research Council (j.nos. 512-9091, 512-10210).
REFERENCES
1. Fraser D. R. and Kodicek E.: Unique biosynthesis by kidney of a biologically active vitamin D metabolite. Nature 228 (1970) 764766. 2. Mawer E. B., Backhouse J., Taylor C. M., Lumb G. A. and Stanbury S. W.: Failure of formation of 1,25-dihydroxycholecalciferol in chronic renal insufficiency. Lancet i (1973) 626-628. DeL&a H. F.: Vitamin D metabolism. C/in. Endocr. 7 suppl. (1977) ls-17s. Stanbury S. W.: The role of vitamin D in renal bone disease. C/in. Endocr. 7 suppl. (1977) 25s30s. Brickman A. S., Coburn J. W. and Norman A. W.: Action of 1,2S-dihydroxycholecalciferol, a potent kidney-produced metabolite of vitamin D3 in uremic man. New Engl. J. Med. 287 (1972) 891-895. R. G., Russell R. G. G., Ledingham 6. Hendersen J. G. G., Smith R., Oliver D. O., Walton R. J., Small D. G., Preston C., Warner G. T. and Norman A. W.: Effects of l,25-dihydroxycholecalciferol on calcium absorption, muscle weakness, and bone disease in chronic renal failure. Lancet i (I 974) 379-384. I. Pierides A. M., Ellis H. A., Simpson W., Cook D. and Kerr D. N. S.: The effect of lee-hydroxyvitamin D3 in predialysis renal bone disease. C/in. Endocr. 7 suppl (1977) 109~116s. M. S.: Bone 8. Melsen F., Nielsen H. E. and Christensen histomorphometry in patients with chronic renal failure: effect of la-hydroxyvitamin D,. C/in. Endocr. 7 suppl (1977) 39+44s. 9. Tougaard L., Sorensen E., Brschner-Mortensen J., Christensen M. S., Rsdbro P. and Sorensen A. W. S.: Controlled trial of la-hydroxycholecalciferol in chronic renal failure. Lancer 1 (1976) 1044-1047.
Chronic renal failure and vitamin D metabolites 10. Christiansen C., Rsdbro P., Christensen M. S., Hartnack B. and Transbel I.: Deterioration of renal function during treatment of chronic renal failure with 1,25-dihydroxycholecalciferol. Lancet ii (1978) 700703. 11. Christiansen C., Redbro P. and Jensen H.: Bone minera1 content in the forearm measured by photon absorptiometry: principles and reliability. Stand. J. c/in. Lab. Invest. 35 (1975) 323-330. 12. Melsen F., Melsen B. and Mosekilde L.: An evaluation of the quantitative parameters applied in bone histology. Acta path. microbial. stand. Sect. A 86 (1978) 63-69. 13. Melsen F., Melsen B., Mosekilde L. and Bergman S.: Histomorphometric analysis of normal bone from the iliac creast. Acta path. microbial. stand. Sect. A 86 (1978) 7&81. 14. Melsen F. and Mosekilde L.: Tetracycline double labelling of iliac bone in 41 normal adults. Calcif: Tim Res. 26 (1978) 99-102. 15. Shepard P. M., Horst R. L., Jorgensen N. A. and DeLuca H. F.: Determination of vitamin D and its metabolites in plasma from normal and anephric man. Biochem. J. 182 (1979) 55-69.
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16. Kiilerich S., Christiansen C., Christensen M. S. and Nzstoft J.: Zinc metabolism in patients with chronic renal failure during treatment with 1,25-dihydroxycholecalciferol: a controlled therapeutic trial. Clin. Nephrol. 15 (1981) 23-27. 17. Malluche H. H., Ritz E., Lange H. P., Kutschera J., Hodgson M., SeitTert V. and Schoppe W.: Bone histology in incipient and advanced renal failure. Kidney Int. 9 (1976) 355-360. 18. Tanaka Y. and DeLuca H. F.: Stimulation of 24,25_dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3. Science 183 (1974) 1198-1200. 19. Christiansen C., Rerdbro P., Christensen M. S. and Hartnack B.: Is 1,25-dihydroxy-cholecalciferol harmful to renal function in patients with chronic renal failure? Clin. Endocr. 15 (1981) 229-236. 20. Fischer J. A. and Binswamzer U.: 1.2%dihvdroxvchole. < calciferol in dialysed patients with clinically asymptomatic renal osteodystrophy. A controlled study. Cow. Nephrol. 18 (1980) 82-91. 21. Malluche H. H., Goldstein D. A. and Massry S. G.: Effects of 6 months therapy with 1,25(OH),D, on bone disease of dialysis patients. Contr. Nephrol. 18 (1980) 9&104.