Relationships of plasma total homocysteine (HCY), folates and vitamin B12 levels to vertebral fracture and bone mineral density in Moroccan healthy postmenopausal women
Aissam El Maataoui1,&, Lamiae Ennefah2, Aziza Mounach3, Hamza Toufik3, Sellama Nadifi4, Abdellah El Maghraoui3, Zahra Ouzzif2
1Faculty of Medicine and Pharmacy, Biochemistry Department, Agadir, Morocco, 2Mohamed V University, Faculty of Medicine and Pharmacy, Military Hospital, Biochemistry Department, Rabat, Morocco, 3Mohamed V University, Faculty of Medicine and Pharmacy, Military Hospital, Rheumatology Department, Rabat, Morocco, 4Hassan II University, Faculty of Medicine and Pharmacy, Casablanca, Morocco
Aissam El Maataoui, Faculty of Medicine And Pharmacy, Biochemistry Department, Agadir, Morocco
a potential role of Homocysteine (HCY) in bone metabolism has been considered from the observation of high prevalence of osteoporosis in subjects with Homocystinuria about 50 years ago. But the mechanism linking the increased level of HCY to increased fracture risk is not clear. The objective of this study was to investigate this possible relationship between vertebral fractures and HCY level in Moroccan postmenopausal women.
one hundred and twenty-two healthy postmenopausal women gave their informed consent to participate in this cross-sectional study. Women were recruited through advertisements and mouth to ear between January 2017 and May 2017. Bone mineral density was determined by a Lunar Prodigy Vision DXA system. Vertebral fracture assessment image was inspected visually by 2 clinicians.
we found that a high level of HCYor low levels of vitamin B12 and folates are not associated to the bone mineral density and are not risk factors for vertebral fractures in healthy postmenopausal women. Whereas, the presence of vertebral fracture was associated to the number of years since menopause and to the OC level. Probably this is due to the young age of the patients involved in this study. We also showed that high level of HCY is associated with the number of years since menopause and not age for women.
we found that a high level of HCY or low levels of vitamin vitamin B12 and folates are not associated to the bone mineral density and are not risk factors for vertebral fracture in healthy Moroccan postmenopausal women.
Several factors are known to affect bone metabolism and to increase
the risk of fracture, one of them is the high level of HCY .
Several studies in postmenopausal women has found an association between
increased plasma concentrations of HCY and low bone mineral density (BMD)
[2-4], while other studies showed no significant association
[5-8]. For the fracture risk, some reports in older
persons have shown an association between elevated plasma HCY and fracture
risk [5, 8-10].
A potential role of HCY in bone metabolism has been considered from the observation
of high prevalence of osteoporosis in subjects with homocystinuria about
50 years ago, Homocystinuria is a genetic disorder caused by mutation of
the cystathionine beta-synthase gene . But, the
mechanism has not yet been elucidated. It has been shown in vitro that high
concentration of HCY decreases the secretion of osteocalcin in preosteoblastic
cells but enhances the secretion of osteopentin [12, 13].
Another mechanism is the increased osteoclast activity associated to the
high level of HCY concentration.In fact, in-vitro study showed that the
increased concentrations of HCY inhibit the activity of lysyloxidase, the
enzyme involved in crosslinking of collagen [14-17],
and interference in cross link formation would cause an altered bone matrix,
resulting in more fragile bone [14-17]. The main objective
of this study was to investigate the relationship between vertebral fracture
and HCY level in Moroccan postmenopausal women.
one hundred and twenty-two healthy postmenopausal women gave their informed consent
to participate in this cross-sectional study. Women were recruited through advertisements
and mouth to ear between January 2017 and May 2017. The procedures of the study
were in accordance with the Declaration of Helsinki, and the Ethics Committee
of the Faculty of Medicine and Pharmacy of Rabat approval was obtained for the
study. Original inclusion criteria were no previous osteoporotic fracture based
on patient record, 24 months of amenorrhea and no previous use of hormone replacement
therapy. Women with liver or renal disease, endocrine or metabolic abnormalities,
and receiving medicine known to influence bone mineralization or levels of HCY
were excluded. Each subject completed a standardized questionnaire designed to
putative risk factors of osteoporosis.
bone mineral density was determined by a Lunar ProdigyVision DXA system (Lunar
Corp., Madison, WI). TheDXA scans were obtained by standard procedures supplied
the manufacturer for scanning and analysis. All BMD measurements were carried
out by experienced technicians. Daily quality control was carried out by measurement
a Lunar phantom. At the time of the study, phantom measurements showed stable
results. The phantom precision expressed as the coefficient of variation percentage
was 0.08. Moreover, reproducibility has been assessed in clinical practice and
showed a smallest detectable difference of 0.04 g/cm2
(spine) and 0.02
(hips). Patient's BMD was measured at the lumbar spine (anteroposterior projectionat
L1-L4) and at the femurs (i.e., femoral neck,trochanter, and total hip). The
WHO classification system was applied, defining osteoporosis as T-score ≤ 2.5
and osteopenia as -2.5 < T-score < -1. Study participants were categorized by
the lowest T-score of the L1eL4 lumbar spine, femur neck, or total femur. VFA
(Vertebral fracture assessment) was classified using a combination of a semi
quantitative (SQ) approach by Genant et al
. and morphometry in the following
manner: each VFA image was inspected visually by 2 clinicians to decide whether
it contained a fracture inany of the vertebrae visualized. Each vertebra that
was judged as fractured by visual inspection by any of the investigators was
measured using built-in morphometry and assigneda grade based on Genant's SQ
1 (mild) fracture is a reduction in vertebral height of 20 to 25%, grade 2 (moderate)
a reduction of 26 to 40%, and grade 3 (severe) a reduction of more than 40%.
all blood samples were collected under fasting conditions on the same day of
acquisition of the VFA images. Blood samples for plasma HCY, folates, osteocalcin,
vitaminB12, and serum parathyroid hormone (PTH) were taken between 8 and 9 am,
in the fasting state, placed on ice, centrifuged within 1h, and the separated
plasma was then immediately stored in 2 different tubes at -80°C until assayed.
These were measured using commercially available kits on the Architect (Abbott
). Plasma HCY was analyzed by commercially available immune-nephelometry
kits with BN Prospec (Siemens healthcare diagnostics®
). The assay
intra and inter-assay CVs of 4.2% and 6.1%, respectively.The Architect 25(OH)D
assay showed excellent precision with a total Coefficient of variance (CV%) < 5%
at the concentrations of quality control material analysed. In the present study,
25(OH) D values of ≤ 20 ng/ml were defined as vitamin D insufficiency and
of ≤ 10 ng/ml as vitamin D deficiency.
results are presented as mean ± standard deviation (SD). To compare patients
with and without VFs Student test was used. To compare patients according to
the quartiles of HCY levels and according to the BMD, analysis of variance was
used. Correlations between different variables were assessed using the non-parametric
Spearman test. Stepwise multiple regression analysis was used to determine the
predictors of BMD. Potential risk factors were entered in a stepwise conditional
binary regression analysis, and the resulted odds ratios (ORs) with 95% confidence
intervals (CIs) were reported. The level of significance was taken as p ≤ 0.05.
Excel 2007 (Microsoft Corp., Redmond, WA) and SPSS 15.0 (SPSS Inc., Chicago,
IL) were used for statistical analysis.
Ethics Committee of the Faculty of Medicine and Pharmacy of Rabat approval was obtained for the study
In this cohort of 122 postmenopausal women, the mean ± SD age, years since
menopause, Body mass index (BMI) and the number of pregnancies showed
a significant difference between the groups of women with normal
bone mineral density (BMD), osteopenia and osteoporosis. While no
significant difference was shown between
this groups for
level, parathormone and Folates (Table
Among the 122 women, 23 (18.85%) had densitometric osteoporosis. Vertebral fracture
(VF) was identified using vertebral fracture assessment (VFA)
in 17 (13.93%) patients. Comparison of patients according
to VF showed a significant difference only for the PTH (p
= 0.003) and the osteocalcin (p = 0.02) (Table
Comparison of patients according to quartiles of HCY levels showed that women
in the highest quartile had a lower level of B12 and higher level
of PTH and a high number of years since menopause (Table
3). In addition to that, a high positive correlation was found between
the number of years since menopause and HCY levels too (Table
The study showed aweak correlation between the T-score at the total hip and the
HCY levels. The most important association is the high positive correlation
found between the HCY levels and the PTH levels. Also, significant
correlations were found between B12, age in years and HC (Table
Multiple regression analysis presented in Table
5 showed that age and B12 were the main predictors of BMD at the total
hip, whereas the main predictor of BMD at the lumbar spine was the age. Stepwise
regression analysis showed that presence of VF was independently related to osteocalcin
and the number of years of menopause (Table
This study showed that HCY, vitamin B12 and folates levels are not
associated to the BMD and are not risk factors for VF in healthy
postmenopausal women.Whereas, the presence of VF was associated
to the number of years
since menopause and to the OC level. We also showed in this study
that high level of HCY is associated with the number of years since
age for women. Patients with VFs compared with those without VF
had significantly higher levels of PTH and OC. Also, We showed
that presence of VF was independently
related to OC level and the number of years of menopause. Several
studies confirmed our results, thereis no or only a weak relation
between HCY and
BMD. In fact, BMD does not reflect the current status of bone metabolism
and presents only poor information about themicroarchitecture of
the bone matrix.
The link between high level of HCY and fracture risk cannot be
attributed to a reduced bone mineralisation [13, 18-20].
We found that high level of HCY was associated to high level
of OC, and Avbersek-Luznik et al reported that increased levels of
bone formation markers are associated with a significantly greater
rate of bone
loss in postmenopausal women .
In this study, The PTH level is highly and significantly elevated in women with
VF and HCY level was positively correlated to the Parathyroid Hormone
(PTH) level. This high PTH levels may indicate increased bone turnover
high level of HCY. In fact, the PTH level is increased to maintain
calcium homeostasis over greater bone turnover .
We did not show a significant association between the plasma HCY level and fracture
risk. In fact, the presence of vertebral fractures was associated
to the OC level and the number of years since menopause in women
in our study.
Previous studies investigating the fracture risk have yielded contradictory
results. In fact, some studies did not show a significantassociation
between the plasma HCY level and fracture risk [22, 23].
But other studies found a significant positiverelation between
HCY plasma levels and fracturerisk especially at the hip [5, 12, 24, 25].
These discrepancies in the results are due to difference in mean
ages of women involved in these studies (from 57 to 70 year-old).
To date, the mechanism
linking HCY to increased fracture risk have not yet been clarified.
B12 and folates are the main determinants in the metabolism of
HCY. Some studies
trials have found that supplementation of folic acid (0.5 - 5 mg
day-1) has resulted in reducing the levels of HCY in blood up to
25% and the co-supplementation
of folic acid and Vitamin B12 (0.5-5 mg day-1 and 500 mcg day-1,
respectively) provided a further reduction of 7% with a decrease
in serum total HCY by
32% . Also, high concentrations of HCY and low
levels of B12 and folates, have been associated with low BMD and
a higher risk of fractures in the elderly . Some
in vitro studies have found that high concentrations of HCY increased
by increasing osteoclast activity [14, 27].
Others in vitro studies have shown that elevated concentrations
of HCY inhibit the activity of the lysyloxydase (involved in cross-linking
of the collagen)
and consequently would lead to an altered bone matrix and enhance
bone fragility [7-9]. In another meta-analysis of observational
studies, structural deterioration of bone tissue have been associated
HCY levels and low vitamin B12 and folates levels, and people with
hip fractures have severe deficiency in folates .
In this study, we showed that in postmenopausal women the number
of years since
menopause and not age reported in previousstudies is the major
determinant of the fracture risk. Our study has strengths and limitations.
All of DXA
measurements were conducted with a single bone densitometerand
all of biochemical exams were done in a single biochemistry laboratory,
with very careful quality
controls in place. The assessment of fracture was carefully conducted
using standard procedures of acquisition and standard reading of
all VFAs. All the morphometric
assessments were made by 2 experienced investigators after training
sessions and a previous global visualization. The main limitations
lie in the cross-sectional
nature of the study and the procedures used to select subjects,
who were all volunteers and ambulatory.
Finally, it is not clear the effect of high HCY level on bone, some authors report a relationship between HCY level and bone, but others find no association. In addition to that, it is difficult to demonstrate whether this is related to HCY level or to the vitamins which are required of its metabolism such as B12, B6, folates.
What is known about this topic
- Several studies in postmenopausal women had found an association between increased plasma concentrations of HCY and low bone mineral density (BMD);
- Other studies showed no significant association;
- For the fracture risk, some reports in older persons have shown an association between elevated plasma HCY and fracture risk.
What this study adds
- First study in the north African population on the association between HCY, vitamin B12, folates levels and vertebral fractures;
- Hcy, vitamin B12 and folates levels are not associated to the BMD and are not risk factors for VF in healthy postmenopausal women;
- We showed that presence of VF was independently related to OC level and the number of years of menopause; patients with VFs compared with those without VF had significantly higher level of PTH and OC were observed among.
The authors declare no competing interests.
Aissam El Maataoui and Zahra Ouzzif contributed to study design,
data analysis/interpretation and in drafting or critically revising
the manuscript. Lamiae Ennefah, Aziza Mounach, Abdellah El Maghraoui
were involved in study
design, results interpretation, and in drafting or critically revising
the manuscript. All authors read and approved the final manuscript.
Table 1: demographic and measured analytes according to BMD status (Mean ±SD) (n = 122)
Table 2: comparison between patients according to vertebral fractures (Mean ±SD) (n = 122)
Table 3: comparison between patients according to the quartiles of HCY (Mean ±SD) (n = 122)
correlations between BMD and biological parameters (n = 122)
multiple regression analysis of the predictors of the bone mineral density at
the lumbar spine and the total hip (n = 122)
regression logistic analysis with the presence of vertebral fracture as the dependent
variable (n = 122)
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