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Second Department of Internal Medicine, Osaka City University Medical School, 1–4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan;
*
Institute of Sumitomo Pharmaceutical Company, Osaka, Japan; and
Kobe Pharmaceutical College, 4–19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe 658, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • albumin • protein deficiency • hyperparathyroidism • osteocalcin • 1,25-dihydroxyvitamin D • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Albright and Reifenstein 1948
). Serum
albumin correlates significantly with skinfold and body mass index
(Thiebaud et al. 1997
), providing a valuable biochemical
marker for nutritional state (Craig 1987
, Laccy 1991
). It has also been shown that serum albumin correlates
significantly with age-adjusted bone mineral density
(BMD)2
(Rico et al. 1992
, Thiebaud et al. 1997
),
whereas low serum albumin was the strongest independent variable
correlated with hip fractures (Punnonen et al. 1986
,
Thiebaud et al. 1997
). In fact, elderly persons who have
osteoporotic fracture are often undernourished as evidenced by
hypoalbuminemia (Cooper et al. 1989
, Rico et al. 1992
, Thiebaud et al. 1997
). Albumin also plays
an important role as a major calcium (Ca)-binding protein in serum
(Payne et al. 1979
). Therefore, it should have more
direct effect on bone metabolism; the reduction in serum albumin
results in an increase in free Ca (Ladensen et al. 1979
), which may suppress bone turnover by inhibiting secretion
of parathyroid hormone (PTH).
It was reported that a pair of siblings with analbuminemia showed
severe juvenile osteoporosis; in these subjects, human serum albumin
replacement therapy prevented the progression of osteoporosis
(Kallee 1996
). Although they developed
hyperparathyroidism, the exact mechanism for the development of
osteoporosis is yet to be determined.
Nagase analbuminemic (NA) rats, which were established from
Sprague-Dawley (SD) rats (Nagase et al. 1979
), lack
serum albumin due to a block of albumin mRNA splicing by a 7-bp
deletion in intron HI of the albumin gene (Esumi et al. 1982
), and thus may provide a relevant animal model for the
investigation of the effect of albumin deficiency on bone metabolism.
We investigated the effect of albumin deficiency and the resulting compensatory increase of protein synthesis on bone metabolism by comparing the basal state of bone metabolism and its responses to ovariectomy (ovx) in NA and control SD rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female 9-wk-old NA rats and female SD rats, which served as a control
group, were purchased from Keari Co. (Osaka, Japan). Rats were caged
individually with free access to food and water, as previously
described (Inaba et al. 1999
). They were maintained on a
12-h light:dark cycle and fed a nonpurified diet (CE Diet, Clea, Japan)
containing 24.8% protein, 1.25% Ca and 1.06% inorganic phosphate
(Pi). The experiment started when the rats were 10 wk old, following a
7-d acclimation period. All animal procedures were approved by the
Institute’s Animal Care Committee. NA rats (n = 70) and SD rats (n = 70) were divided into two
groups, one group undergoing a sham operation (sham) and the other
group undergoing ovx. Under anesthesia with ether, bilateral ovx was
performed in 12-wk-old NA (n = 35) and SD rats
(n = 35) by using a dorsal approach with a small
single midline dorsal skin incision. The other NA and SD rats
(n = 21 each) were subjected to a sham operation by
exposing but not removing the ovaries. Success of ovx was confirmed at
necropsy by failure to detect ovarian tissue and by the observation of
marked atrophy of uterine horns.
The rats were weighed biweekly and checked for general health and food intake. To facilitate 24-h collection of urine, the rats were placed in individual metabolic cages with free access to the same food and water. After being deprived of food overnight, but with free access to water, the rats (n = 7/treatment) were killed by aortic puncture at the indicated times. The blood obtained was centrifuged at 1,200 g for 10 min and the serum was stored at -80°C until analysis.
Biochemical variables.
Serum levels of total protein, albumin, 
1-, 2-, ß- and 
-globulins, glucose, cholesterol, triglyceride,
serum urea nitrogen (SUN), creatinine (Cr), alkaline phosphatase (ALP),
total Ca, and Pi were measured with a Hitachi model 7450 autoanalyzer
(Hitachi, Tokyo, Japan) and ionized Ca with NOVA CRT-8 (NOVA
Biomedical, Tokyo, Japan). Urinary Ca and Cr were also measured with
the autoanalyzer, and urinary excretion of Ca was corrected for that of
Cr. Serum PTH was measured by immunoradiometric assay (Allegro Intact
PTH, Nichols Institute, San Juan Capistrano, CA), which measures only
active intact PTH but not degradation products resulting from its
cleavage (Finch et al. 1992
). Serum osteocalcin (OC),
also known as bone Gla-protein, was measured with a commercial RIA
kit (Yamasa Shoyu, Chosi, Japan), using rat OC as a standard
(Inaba et al. 1999
, Nakatsuka et al. 1991
).
Assay of vitamin D metabolites and vitamin D–binding protein (DBP).
Serum levels of vitamin D metabolites and DBP were determined at 3 wk
after ovx. Vitamin D metabolites were assayed for serum levels by
Mitsubishi Kagaku Bio-Clinical Laboratories (Tokyo, Japan), as
described previously (Inaba et al. 1997
). Samples were
dried with nitrogen and then resuspended in 300 µL of
methanol/isopropanol/n-hexane (1:6:93, v/v/v) just
before injection. Vitamin D metabolites were separated by HPLC
(Inaba et al. 1991
and 1997
) with a 0.46 x 30.0 cm
APS-Hypersil NH2 column (Shandon, Cheshire, UK).
Methanol/isopropanol/n-hexane (1:6:93) comprised the
elution solvent, which was passed through a 0.5-µm
filter (Millipore, Medford, MA) just before use. The column was eluted
at a flow rate of 2 mL/min and 4-mL fractions were collected.
Absorbance was measured at a wavelength of 264 nm by a JASCO 875 UV
(Japan Spectroscopic, Tokyo, Japan). After vitamin D metabolites were
separated, levels of 25-hydroxyvitamin D (25-OH-D) and
24,25-dihydroxyvitamin D [24,25-(OH)2D] were measured by
an established competitive protein binding assay using vitamin
D–deficient rat serum. Levels of 1,25-dihydroxyvitamin D
[1,25-(OH)2D] were measured by radioreceptor assay using
vitamin D receptor extract from bovine thymus (Yamasa Shoyu, Choshi,
Japan), as described previously (Inaba and DeLuca. 1989
,
Inaba et al. 1997
). Serum DBP, determined by rocket
immunoelectrophoresis, was expressed relative to that of a control
sample (Masuda et al. 1989
).
Bone densitometry.
BMD was measured in the proximal tibia with dual energy X-ray
absorptiometry (DCS-600 Aloka, Tokyo) every 2 wk until the age of 18 wk
(Oikawa et al. 1999
).
Statistical analysis.
Values are expressed as means ± SD unless otherwise indicated. Statistical analysis was performed by two-way ANOVA, followed by Fisher’s paired least-significant difference (PLSD) test. Time effect was tested by ANOVA for repeated measurement and post-hoc tests. The assessment of differences between NA and SD rats was performed by Student’s t test. A level of P < 0.05 was regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A typical electrophoretic analysis of serum protein in NA rats
indicated a serum albumin concentration of only 1.8 g/L (total protein,
63.0 g/L; albumin, 2.8%; 1-globulin, 54.7%;
2-globulin, 12.4%; ß-globulin, 23.3%; and
-globulin, 6.8%) with an albumin/globulin (A/G) ratio of 0.03, in
contrast to 36.4 g/L (total protein, 68.0 g/L; albumin, 53.5%;
1-globulin, 22.3%; 2-globulin, 6.3%; ß-globulin, 13.7%; and -globulin,
4.2%) with an A/G ratio of 1.15 in SD rats. Body weight did not differ
between NA and SD rats at the age of 12 wk (Table 1
). There were no differences in serum concentrations of total Ca, Pi and
Mg, but serum ionized Ca was significantly lower in NA rats than in SD
rats. Furthermore, NA rats had significantly greater serum
concentrations of intact PTH and OC and urinary excretion of Ca than SD
rats. No significant difference in SUN or serum Cr concentrations was
found between NA and SD rats, negating the possibility of uremic
secondary hyperparathyroidism in NA rats. NA rats also had
significantly greater serum concentrations of total cholesterol and
triglycerides compared with SD rats.
|
Body weights of SD and NA rats in the sham group increased
significantly by 34 and 28%, respectively, during the 8-wk
experimental period (Fig. 1
). Weight gain was significantly greater in the ovx groups than in the
sham groups.
|
Serum total Ca did not differ significantly between NA and SD rats in
either the sham or ovx groups (Fig. 2
). However, although serum Pi did not differ significantly between NA
and SD rats before ovx or sham operation, serum Pi in NA rats became
greater than that in SD rats after surgery. Significantly greater
urinary Ca excretion was observed in NA rats compared with SD rats,
both at the time of ovx and after 3 wk [urinary Ca/Cr
(µmol/mmol), 1.54 ± 0.31 vs. 1.12 ± 0.34 at
the time of ovx, P < 0.05; 1.40 ± 0.17 vs. 0.92
± 0.45 at 3 wk after ovx, P < 0.05]. Serum PTH
(Fig. 3A
) and OC (Fig. 3B
) levels were significantly higher in NA
rats than in SD rats at the time of surgery, and 1 and 3 wk afterwards.
Ovx induced a significant increase in serum PTH and OC in NA rats but
not in SD rats.
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Serum 1,25-(OH)2D levels, determined at 3 wk
after ovx, were significantly greater in ovx NA rats by ~51 and 96%
compared with sham NA rats and ovx SD rats, respectively
(Fig. 4A
). In contrast, serum 24,25-(OH)2D and 25-OH-D
were significantly higher in ovx SD rats than in either ovx NA rats or
sham SD rats. Even after serum vitamin D concentrations were corrected
for DBP, the ratios of 1,25-(OH)2D/DBP,
24,25-(OH)2D/DBP and 25-OH-D/DBP, which are
considered to be indices of free vitamin D metabolites, showed
essentially the same patterns (Fig. 4B
).
|
BMD in proximal tibia did not differ significantly between NA and SD
rats at the time of ovx (Fig. 5
). Although BMD increased steadily over time in sham NA and SD rats,
there was no significant difference between the two groups. However,
ovx ablated the time-dependent increase of BMD in SD rats. Of great
interest was that in ovx NA rats, BMD decreased significantly by 2 wk
after ovx. Ovx NA and SD rats had significantly lower BMD at all time
points after surgery than their respective sham-operated rats.
Furthermore, ovx NA rats had significantly lower BMD than ovx SD rats
at all times after ovx.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Because NA rats genetically lack the ability to synthesize albumin, a
compensatory increase in the synthesis of proteins and lipids appears
to cause an increase in serum cholesterol and triglyceride
concentrations (Takahashi et al. 1983
), as described in
nephrotic patients (Kaysen 1992
). The plasma protein
concentration is nearly normal in NA rats because albumin is replaced
by several high-molecular-weight proteins, including
2-macroglobulin and
1-inhibitor 3 as in rats with nephrotic
syndrome (Stevenson et al. 1998
).
Although serum albumin is a valuable biochemical marker of nutritional
state, albumin should have a more direct effect on bone metabolism
because of its role as a major Ca-binding protein; hypoalbuminemia
should result in an increase in free Ca, which may suppress bone
turnover by inhibiting PTH secretion from the parathyroid gland.
However, because serum total Ca levels did not differ significantly
between NA and SD rats, NA rats have other substances acting as
Ca-binding proteins. Serum levels of ionized Ca at the time of ovx
were significantly lower in NA rats than in SD rats, although serum
total Ca and Pi did not differ significantly between NA and SD rats,
suggesting that the major Ca-binding substance in the serum of NA
rats might have higher affinity for Ca than albumin. A significant
reduction in serum ionized Ca in NA rats may contribute in part to the
enhancement of bone turnover by inducing hyperparathyroidism.
Furthermore, a significant increase in conversion of 25-OH-D to
1,25-(OH)2D, in association with a significant
reciprocal suppression of 24,25-(OH)2D conversion
in NA ovx rats compared with SD ovx rats, clearly supported the
hypothesis that ovx paradoxically augmented hyperparathyroidism in NA
rats as evidenced by increased synthesis of
1,25-(OH)2D. Vitamin D binds mainly to DBP and
albumin (Bikle and Gee 1989
). DBP, which has striking
homology with albumin (Gibbs and Dugaiczyk 1987
), is a
nutritional marker (Polberger et al. 1990
) like albumin.
Therefore, because it was possible that serum DBP level may be
different in NA and SD rats, a free index was calculated using
DBP-adjusted serum levels of vitamin D metabolites (Bikle and Gee 1989
, Nyomba et al. 1989
,
Woloszczzuk 1985
). However, even after normalization of
serum vitamin D metabolites to DBP, an activation step of vitamin D
from 25-OH-D to 1,25-(OH)2D remained
significantly enhanced in NA rats (Fig. 4B
). Although the
development of hyperparathyroidism in NA rats may be explained in part
by a significant reduction in serum ionized Ca, it cannot be the sole
factor involved. Serum Pi did not differ significantly between NA and
SD rats at the time of ovx when NA rats had a greater serum
concentration of PTH. Because PTH has a strong phosphaturic action, Pi
entry into the circulation likely was greater in NA rats. Furthermore,
serum Pi levels became greater in NA rats than in SD rats thereafter in
spite of higher PTH levels (Fig. 2B
), clearly indicating the
occurrence of increased Pi load as a major contributing factor to the
development of hyperparathyroidism in NA rats (Fig. 3A
). One
possibility that would explain this is demonstrated in nephrotic
patients, in whom it has been shown that intestinal Pi absorption is
not reduced despite a significant reduction in Ca absorption
(Farrington et al. 1983
). The same phenomenon may occur
in NA rats, thus contributing to the development of hyperphosphatemia.
Alternatively, in the absence of albumin, the transport of
albumin-bound substances, including Ca, from the blood plasma into
extravascular space and vice versa is severely disturbed, which may
cause hyperparathyroidism in NA rats.
Contrary to previous results indicating the suppressive effect of ovx
on parathyroid function (Pioli et al. 1992
), ovx
enhanced hyperparathyroidism in NA rats. Because estrogen has a direct
effect on suppression of bone resorption and therefore, bone resorption
is enhanced by estrogen deficiency (Parfitt 1988
,
Wronski et al. 1988
), ovx was expected to suppress serum
PTH by increasing bone-derived Ca entry into the blood stream.
Unexpectedly, NA rats showed a significant increase of serum PTH
following ovx. Although plasma lipid levels are expected to decrease by
estrogen replacement as in postmenopausal women (Heikkien et al. 1999
), a paradoxical rise in plasma lipid level was reported in
ovx NA rats given estrogen replacement (Takahashi et al. 1983
), suggesting that NA rats respond paradoxically to
estrogen deficiency not only in Ca metabolism but also in lipid
metabolism.
In summary, the present study demonstrated that, in NA rats with analbuminemia and a compensatory increase in protein synthesis, development of hyperparathyroidism may be due to an increased Pi load and a lower ionized Ca concentration. Moreover, the augmentation of hyperparathyroidism observed in NA rats due to ovx could result in the greater reduction of BMD in ovx NA rats than in ovx SD rats. However, in the hypoalbuminemia resulting from impaired synthesis of generalized protein in liver such as liver cirrhosis, the extent of the influence of hypoalbuminemia on bone metabolism is not known from this study.
In conclusion, hypoalbuminemia may cause hyperparathyroidism by increasing the level of serum Pi and suppressing ionized Ca concentration. Hence, the NA rats readily lose bone following ovx.
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FOOTNOTES |
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Manuscript received August 26, 1999. Initial review completed September 27, 1999. Revision accepted February 8, 2000.
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