Sterol Metabolism in Ovariectomized Rats: Potential Roles of Campesterol and 4β‐Hydroxycholesterol in Osteoporosis

ABSTRACT

Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue. Lipid metabolism disorders may contribute to osteoporosis in postmenopausal women. Phytosterols and oxysterols (OC) are involved in lipid metabolism. However, their roles in bone metabolism and in the development of osteoporosis remain unclear. This study used ovariectomized (OVX) rats, commonly used to study osteoporosis, to analyze the levels of sterols, including phytosterols, cholesterol, and oxysterols in the humerus, femur, femoral bone marrow, blood, and liver. Sterol levels were measured through gas chromatography. We observed that campesterol and cholesterol exhibited similar patterns of change in the blood, liver, and bone marrow. Conversely, cholesterol and campesterol levels in the femur did not change significantly, while bone marrow cholesterol levels decreased. The similar changes in campesterol and cholesterol levels suggest that these sterols are derived from the blood and taken up by the bone marrow. Significant increases in 4β‐hydroxycholesterol were found in the humerus, plasma, and liver of OVX rats. The ratio of 4β‐hydroxycholesterol to cholesterol in the bone marrow was significantly increased in OVX rats. These findings suggest that 4β‐hydroxycholesterol is associated with OVX‐induced osteoporosis and contributes to the decline in bone density.

Abbreviations

25‐OH

25‐hydroxycholesterol

27‐OH

27‐hydroxycholesterol

4β‐OH

4β‐hydroxycholesterol

5α‐OH, 6‐keto

5α‐hydroxy‐6‐ketocholestanol

6‐keto

6‐ketocholestanol

7‐keto

7‐ketocholesterol

7α‐OH

7α‐hydroxycholesterol

7β‐OH

7β‐hydroxycholesterol

Chol

cholesterol

GC

gas chromatography

OC

oxysterol

OVX

ovariectomized

SD

Sprague–Dawley

α‐epoxy

5α6α‐epoxycholesterol

β‐epoxy

5β6β‐epoxycholesterol

β‐triol

β‐cholestanetriol

1. Introduction

Osteoporosis is characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in increased bone fragility and a consequent rise in fracture risk (Hu et al. 2018). The etiology of osteoporosis includes physical inactivity, smoking, and alcohol consumption, and deficiencies in vitamin D, calcium, and estrogen in postmenopausal women (Lu and Tian 2023). Moreover, it is frequently observed in conjunction with lifestyle‐related conditions such as dyslipidemia.

Lipid metabolism disorders may contribute to postmenopausal osteoporosis. Studies using ovariectomized (OVX) mice (Li et al. 2024), a model for postmenopausal osteoporosis, as well as clinical studies in humans (Li et al. 2020; Shen et al. 2012), have reported that increased bone marrow adiposity, an indicator of dysregulated lipid metabolism, contributes to bone loss. Several studies have indicated a relationship between reduced bone mineral density (BMD) and abnormal cholesterol metabolism. In postmenopausal women, serum cholesterol levels negatively correlate with BMD (Makovey et al. 2009). Furthermore, in vitro, extracellular cholesterol significantly enhances osteoclast activity in osteoclasts (Hada et al. 2012). Due to the low expression of HMG‐CoA reductase in osteoclasts (Bergstrom et al. 2000), which is the rate‐limiting enzyme in intracellular cholesterol biosynthesis, their differentiation and activity are highly dependent on serum lipoprotein‐derived cholesterol (Luegmayr et al. 2004). The balance among synthesis, efflux, and uptake determines the availability of intracellular cholesterol. Regarding synthesis, as HMG‐CoA reductase inhibitors, statins exert bone‐protective effects by increasing BMD and reducing fracture risk in postmenopausal women (Edwards et al. 2000; Uzzan et al. 2007). Statins also increase bone morphogenetic protein‐2 (BMP‐2) mRNA in osteoblasts, thereby promoting osteoblast differentiation (Ghosh‐Choudhury et al. 2007). On the other hand, cholesterol efflux is facilitated by activation of liver X receptor (LXR) (Griffiths and Wang 2022). Oxysterols (OC, Oxidized cholesterol derivatives) act as LXR ligands with the monkey‐derived kidney cell line CV‐1 (Lehmann et al. 1997), suggesting that OC molecules may also enhance cholesterol efflux via LXR activation. A previous study reported elevated plasma OC levels in Sprague–Dawley (SD) rats fed a high‐cholesterol diet (Liu et al. 2018). One of the OCs, 27‐hydroxycholesterol (27‐OH), shows the ability to promote osteoclast differentiation and activity and inhibit osteoblast function in vitro (Nelson et al. 2011), ultimately resulting in bone loss. These findings suggest that abnormalities in the metabolism of cholesterol and OCs may contribute to the development of osteoporosis. However, the characterization of OCs within bone remains insufficient.

Dietary sterols consist of animal‐derived cholesterol and plant‐derived phytosterols. A previous study demonstrated that 12 weeks of oral olive oil administration significantly suppressed OVX‐induced bone loss in 6‐month‐old SD rats (Liu et al. 2014). Given the low content of n‐3 polyunsaturated fatty acids, which are known regulators of bone metabolism, in olive oil (Nawata et al. 2013), it has been suggested that the abundant phytosterols in olive oil may contribute to the regulation of bone metabolism. Phytosterols are abundant in foods such as nuts, seeds, and legumes. Intake of phytosterols has been reported to reduce serum cholesterol levels (Xia et al. 2022). Moreover, a recent study suggests that β‐sitosterol suppressed osteoporosis by regulating the differentiation of bone marrow adipocytes and promoting osteoblast activity (Li et al. 2025). These findings suggest that phytosterols may also play a role in the regulation of bone metabolism. In general, phytosterols exhibit low intestinal absorption rates ranging from 0.04% to 16% in the intestine (Brufau et al. 2008). Due to their structural similarity to cholesterol, phytosterols compete with cholesterol for uptake via the Niemann‐Pick C1‐Like 1 (NPC1L1) transporter (Davis Jr et al. 2004) and are subsequently incorporated into chylomicrons for delivery to the liver. In the liver, phytosterols undergo limited metabolism and are excreted into the bile by ATP‐binding cassette (ABC) transporters, such as ABCG5 and ABCG8 (Brufau et al. 2008). Consequently, plasma phytosterol levels remain lower than the cholesterol level, but they can still exert biological effects on lipid metabolism. However, sterol analysis of bone marrow has not yet been conducted. In this study, we aimed to investigate the relationship between osteoporosis and 19 sterol species, including cholesterol, OCs, and phytosterols. To this end, we utilized an OVX rat model of osteoporosis to quantify the levels of sterol in bone and bone marrow and to identify specific alterations associated with osteoporosis.

2. Method

2.1. Preparation of Osteoporosis Model Rats

This study was conducted with the approval of the Kyushu University Animal Experiment Committee (Approval No: A30‐313‐0) and the President of Kyushu University. Ten‐week‐old female Sprague–Dawley rats (SLC:SD) were obtained from Japan Slc Inc. (Shizuoka, Japan). Rats were housed individually at a controlled temperature of 22°C ± 2°C with a 12‐h light/dark cycle (lights on at 0800 and off at 2000) and handled according to the Animal Experiment Handling Regulations of Kyushu University's Faculty of Agriculture. For 1 week, the rats were pre‐fed with a commercial solid feed (CRF‐1, Oriental Yeast) powdered using a mixer.

Following acclimatization, the rats were randomly assigned to two groups: ovariectomy (OVX) surgery and sham (SHAM) surgery under isoflurane anesthesia. In the OVX group, an incision was made through the skin and muscle layer along the abdomen, the area between the ovaries and uterus was ligated, and the oviduct above the ligature was cut to remove the ovaries. The incision was then closed to induce an estrogen‐deficient state. In the SHAM group, an abdominal incision was made, but no ligation or removal of the ovaries was performed, and the incision was simply sutured. During surgery, sulfamethoxazole (Shionogi & Co., Osaka) was applied to the incision site as an antibiotic, and Betrofar (Meiji Seika, Tokyo) was administered as an analgesic before suturing. Incisions were closed with sutures and secured with Michel clips. Following surgery, the rats were placed on a warming platform until they recovered. Once they resumed spontaneous movement, they were housed individually and allowed a one‐week postoperative period. On surgery day, the rats were given recovery water (25.0 g/L glucose and 4.97 g/L NaCl dissolved in 2.5 L deionized water).

A week post‐surgery, rats received a modified AIN‐76 formula containing 5% olive oil and 0.5% cholesterol (Table 1) from 1700 to 0900 daily for 4 weeks, ensuring consistent intake relative to body weight. Sterol composition in the diet is shown in Table 2. After the feeding period, rats were fasted for 9 h and then euthanized under isoflurane anesthesia by blood collection from the aorta.

TABLE 1.

The composition of experimental diet.

Ingredients	(g/100 g)
Cellulose	5.0
Casein	20.0
Olive oil	5.0
Corn starch	15.0
Sucrose	49.5
Vitamin mix (AIN‐76)	1.0
Mineral mix (AIN‐76)	3.5
DL‐Methionine	0.3
Choline bitertrate	0.2
Cholesterol	0.5
Total	100.0

Note: The diets are formulated according to the AIN‐76 formula.

TABLE 2.

Dietary sterol levels.

	(μg/g diet)
Cholesterol	749 ± 25.4
Campesterol	7.50 ± 0.197
Stigmasterol	0.873 ± 0.003
β‐Sitosterol	9.97 ± 0.607

Note: Measurement was triplicate. Values are means ± S.E.

Blood samples were collected in vacuum blood collection tubes (Venoject Terumo), centrifuged at 1500×g (RS‐205, TOMY) for 20 min at 4°C, and plasma was separated. Plasma samples were treated with 2,6‐Di‐t‐butyl‐p‐cresol (BHT, Nacalai Tesque) and stored at −30°C. For liver analysis, a 0.5 g portion was required for lipid analysis. The humerus was collected for lipid analysis, and the femur for both lipid and bone density analysis. The remaining liver tissue and the 0.5 g portions were frozen in liquid nitrogen and stored at −30°C until analysis.

2.2. Measurement of Femoral Bone Density

The femoral bone density was measured using an x‐ray CT scanner LaTheta LCT‐100 for laboratory animals. The measurement conditions were set as follows: small field of view, high precision rotation speed, 30 slices, and a slice interval of 1 mm. Cross‐sectional images were observed from the proximal femoral epiphysis (the region up to where the medullary cavity appeared), the diaphysis (the slice range where the medullary cavity was visible), and the distal femoral epiphysis (the area from where the medullary cavity was no longer visible to the distal end). Bone density parameters of the femur were analyzed by region. The parameters included total bone density, trabecular bone density, cortical bone density, and trabecular structure density, all expressed in mg/cm³. Additionally, the minimum cross‐sectional second moment of area, representing resistance to bending, and the polar second moment of area, representing resistance to torsion, were measured in mg·cm.

2.3. Lipid Extraction From the Liver and Bone Marrow

Lipids were extracted from the liver and bone marrow using the Folch method (Folch et al. 1957). The frozen femurs, stored at −30°C, were cut at the epiphysis and placed cut‐side down in a 1.5 mL Eppendorf tube. The tubes were centrifuged at 4°C and 2000×g for 5 min. After weighing the collected bone marrow, the entire amount was sampled from the tube for extraction and concentration. Briefly, lipids were extracted using a chloroform/methanol solution (2:1 by volume). The samples were then incubated in a 40°C water bath for 30 min. After evaporating the organic solvent, the final volume was adjusted to 25 mL with hexane.

2.4. Lipid Extraction From the Femur and Humerus Samples

The femur with marrow removed and the humerus with marrow intact were used. The bone weights were recorded before extraction. The bones were placed in a screw‐cap tube, and 3 mL of methanol was added. The mixture was incubated in a 40°C water bath with shaking at 100 rpm for 60 min. Afterward, 6 mL of chloroform was added, and the samples were left to stand overnight at 4°C. The next day, the chloroform‐methanol solutions were dried up under nitrogen gas, and 5 mL of hexane was added. These hexane solutions were then transferred to a 10 mL volumetric flask. The solutions were filled up to 10 mL and used in the experiment just before analysis.

2.5. Lipid Extraction From Plasma Samples

Plasma was spiked with 5 μg 5α‐cholestane (Sigma) as an internal standard. 250 μL of plasma, 0.9 mL of methanol, and 2.8 mL of chloroform were added to a screw‐cap tube in order. The mixture was gently mixed and incubated at 40°C for 30 min. After extraction, 0.8 mL ultrapure water was added and mixed. Then, the samples were centrifuged at 4°C, 1000×g for 5 min. The lower layer was transferred to a new screw‐cap tube, and another 2.8 mL of chloroform was added. Samples were centrifuged again under the same conditions. This step was performed three times. Throughout the procedure, argon gas was used to fill the tube to prevent oxidation. The combined lower layers were dried under nitrogen gas and prepared for saponification.

2.6. Sample Preparation for Derivatization and GC Analysis of Phytosterols, Cholesterol and Pre‐Cholesterols

The sterols sample preparation method is based on our previous research with some modifications (Wang et al. 2024). For the liver, femur bone marrow, humerus, and femur, lipid extracts of 1, 4, 8, and 2 mL, respectively, were sampled into screw‐cap tubes, dried under nitrogen gas, and spiked with 5 μg of 5α‐cholestane (Sigma) as the internal standard. Then, 1 mL of 0.71 M ethanol‐potassium hydroxide (EtOH‐KOH) was added to each sample (plasma, liver, femur bone marrow, humerus, and femur). After filling with argon gas, the samples were heated in a water bath at 65°C for 1 h to saponify. After cooling to room temperature, 0.5 mL of ultrapure water was added. Then, 2 mL of hexane was added, argon gas was filled, and the mixture was vortexed for about 15 s before being centrifuged at 18°C, 1400×g for 10 min. The hexane layer was collected and transferred to a round‐bottom tube. This step was repeated twice, and the collected upper layers were dried under nitrogen gas and filled with argon gas. To each tube, N,O‐bis(trimethylsilyl)trifluoroacetamide with TMCS (BSTFA‐TMCS 99:1) was employed as a derivatizing reagent. 0.3 mL of the reagent was added to each tube, and the samples were immediately vortexed. The derivatization reaction was conducted at 70°C for 60 min to form trimethylsilyl (TMS) derivatives. After nitrogen drying, 1 mL of hexane was added, and the samples were centrifuged at 1400×g for 5 min. The supernatants were transferred to a glass screw cap tube, dried under nitrogen gas, and concentrated to 15 μL. Finally, 2 μL of the sample was injected into both GC–MS and GC‐FID for analysis. GC–MS‐QP2020 (Shimadzu Corporation, Kyoto, Japan) and GC‐2014s (Shimadzu Corporation, Kyoto, Japan) were equipped with SPB‐1 fused‐silica capillary column (L × I.D.: 60 m × 0.25 mm; thickness: 0.25 μm; Supelco, PA, USA). The following temperature program was applied with helium (high purity: 99.9999%, Asahi Oxygen, Fukuoka) as the carrier gas at a flow rate of 1.5 mL/min: 180°C for 1.0 min; from 180°C to 250°C at 20°C/min; and from 250°C to 290°C at 5°C/min and held at 290°C for 37.5 min. The total run time was 50 min. The injector split ratio was 1:10. The ion source was maintained at 300°C, and the interface was maintained at 250°C. The characteristic fragment ions for each precursor of cholesterol, cholesterol, and phytosterol were identified based on our previous research (Table S2). The analysis of the phytosterols, cholesterol precursors, and cholesterol was based on our research described before (Yuan et al. 2021).

2.7. Sample Preparation for Derivatization and GC Analysis of Oxysterols

The oxysterol analysis was adapted from our previous research with some modifications. Lipid extracts were processed to yield around 1000 ng of total oxysterols per sample tube, to which 100 ng of 19‐hydroxycholesterol (Sigma) was spiked with an internal standard. Samples were saponified by adding 4 mL of 1 M EtOH‐KOH and kept at room temperature in the dark overnight. After saponification, 4 mL of ultrapure water and 4 mL of hexane were sequentially added, and the mixture was vortexed for 5 min and then centrifuged at 1500×g for 5 min. The upper layer was collected and transferred to a new tube. This extraction step was repeated to enhance recovery efficiency, and all tubes were filled with argon gas to prevent oxidation. The collected upper layers were dried under nitrogen gas and reconstituted in 1 mL of toluene. The solutions were then applied to Sep‐Pak silica cartridges (Waters, USA) preconditioned with 2 mL of hexane in each solution. The column was washed with 1 mL of hexane, followed by 10 mL of 0.5% isopropanol in hexane. Then, OCs were eluted with 5 mL of 30% isopropanol in hexane. The eluates were dried under nitrogen and derivatized with 0.2 mL of a TMS derivatization reagent [pyridine: hexamethyldisilazane: trimethylchlorosilane = 9:3:1 (v/v/v)] by vortexing gently every 5 min during a 30 min reaction at room temperature. After derivatization, the reagent was removed under nitrogen, and the TMS‐derivatives were re‐dissolved in 1 mL of anhydrous ethyl acetate. The solutions were centrifuged at 1500×g for 5 min, and the supernatants were transferred to new clean tubes for GC–MS analysis. GC–MS was performed on a Shimadzu GC‐2014s instrument (Shimadzu Corporation, Kyoto, Japan) coupled with an Inert Cap 5MS/NP capillary column (L × I.D.: 30 m × 0.25 mm; thickness: 0.25 μm, GL Sciences Inc., Tokyo, Japan), connected to a QP2020 series mass‐selective detector (Shimadzu). The following temperature program was applied with helium (high purity: 99.9999%, Asahi Oxygen, Fukuoka) as the carrier gas at a flow rate of 1.5 mL/min: 180°C for 1 min; ramp 180°C to 250°C at 20°C/min; and ramp 250°C to 290°C at 5°C/min and held at 290°C for 17.50 min. The total run time was 30 min. The injector split ratio was 1:5. The ion source was kept at 300°C, and the interface was kept at 250°C. Calibration curves were made with mixtures of purchased pure OC molecules. m/z values for each OC molecule were represented in Table S3. Quantification of each oxysterol was performed using calibration curves based on the ratio of analyte peak area relative to the internal standard. Compound identities were confirmed by retention time alignment and mass spectral matching with authentic standards as we described before (Shirouchi et al. 2019).

2.8. Statistical Analysis

Regarding sterols analysis for GC, all waveform processing, peak area, and concentration calculations were performed using GC–MS Solution Ver. 1.20 (Shimadzu Corporation) software. Data are expressed as the means ± standard error of the mean (SEM). All data were analyzed with Student's t‐test. Statistical significance was defined as p < 0.05, and a trend was indicated for p < 0.10.

3. Result

3.1. OVX Rats Exhibit Significantly Reduced Bone Mineral Density

Initially, variability was observed in the estrogen deficiency induced by OVX (Table S1). To maximize the evaluation of OVX representation, individual animals were selectively chosen. It is reported that the deficiency of estrogen increased the level of blood cholesterol in rats (Ke et al. 1997). Considering the focus on the relationship between sterol levels and bone mineral density, the sample selection was based on plasma cholesterol levels. Among the 8 ovariectomized rats, the five with the highest plasma cholesterol levels were assigned to the OVX group. Conversely, among the seven rats with SHAM surgery, the five with the lowest plasma cholesterol levels were assigned to the SHAM group. After selection, plasma cholesterol levels and uterine weights in the two groups are presented in Table 3. As presented in Table 4, after selection, OVX rats tended to have lower mean body weight compared with the SHAM group, while food intake, body weight gain, and the white adipose tissue weight were comparable between the SHAM and OVX groups, which indicated that the high level of plasma cholesterol in the OVX group was not caused by hyperphagia or fat mass difference.

TABLE 3.

Uterus weight and plasma cholesterol level (after selection).

	SHAM	OVX	Statistics
Uterus weight (g)	0.724 ± 0.104	0.545 ± 0.042	p = 0.076
Plasma cholesterol level (mg/dL)	117 ± 13.5	191 ± 19.5	p < 0.05

Note: Values are means ± S.E. (n = 5/group). Significant differences between mean values were evaluated by Student's t‐test.

TABLE 4.

Growth parameters (after selection).

	SHAM	OVX	Statistics
Initial body weight (g)	233.2 ± 4.4	220.3 ± 3.8	p = 0.058
Final body weight (g)	273.6 ± 5.1	258.4 ± 5.3	p = 0.071
Body weight gain (g)	40.4 ± 3.4	38.1 ± 2.4	N.S.
Food intake (g/day)	16.2 ± 0.3	15.9 ± 0.1	N.S.
Food efficiency	88.9 ± 6.4	85.5 ± 5.5	N.S.
(mg body weight gain/g total food intake)
Muscle
Femoral muscle	1.467 ± 0.021	1.402 ± 0.051	N.S.
Gastrocnemius muscle	1.300 ± 0.022	1.332 ± 0.050	N.S.
White adipose tissue
Perirenal	0.677 ± 0.039	0.762 ± 0.089	N.S.
Mesenteric	2.126 ± 0.197	2.051 ± 0.148	N.S.
Subcutaneous	4.502 ± 0.216	4.446 ± 0.281	N.S.
Bone
Femur	0.558 ± 0.015	0.563 ± 0.020	N.S.
Humerous	0.266 ± 0.013	0.255 ± 0.008	N.S.

Note: Values are means ± S.E. (n = 5/group). Significant differences between mean values were evaluated by Student's t‐test.

Compared with the SHAM group, the OVX group showed significantly lower cortical bone density, trabecular bone density, total bone density, and femoral bone density (Figure 1a–d). Bone strength indicators, such as the minimum moment of inertia of area and the polar moment of inertia of area, also showed decreasing trends in the OVX group (Figure 1e,f). In addition, the OVX group demonstrated an increase in plasma cholesterol level and a tendency toward reduced uterine weight. The observed reductions in femoral bone density and bone strength indicators in the OVX group compared to the SHAM group further confirm the successful establishment of the OVX model in this study. Accordingly, the 10 selected rats (five per group) were used in the subsequent experiments.

FIGURE 1.

Femoral bone density parameters. (a) Cortical bone density. (b) Cancellous bone density. (c) Trabecular bone density. (d) Whole bone density. (e) Minimum moment of inertia of area. (f) Polar moment of inertia of area. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test.

3.2. Elevated Humoral Cholesterol Levels in OVX Rats

Sterol levels were analyzed in the humeri collected from the rats. In the OVX group, cholesterol levels in the humerus tended to be higher compared to the SHAM group (p = 0.0612, Figure 2a). In contrast, phytosterols such as campesterol and β‐sitosterol showed no significant differences (Figure 2b,c). Subsequently, the femur was analyzed due to its suitability for separating bone marrow and bone tissue, allowing independent assessment of sterol levels in each compartment.

FIGURE 2.

The humerus bone sterols. (a) Cholesterol. (b) Campesterol. (c) β‐sitosterol. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test.

3.3. Ovariectomy Reduces Cholesterol and Campesterol Levels in Femoral Bone Marrow Without Affecting Overall Lipid Content

In the femoral bone, the level of squalene, a precursor for cholesterol synthesis, showed a tendency to increase in the OVX group compared to the SHAM group, while cholesterol levels remained unchanged (Figure 3a). In femoral bone marrow, the levels of cholesterol and campesterol were significantly reduced in the OVX group, while no significant differences were observed in the levels of stigmasterol and β‐sitosterol (Figure 3b). In both bone and marrow, desmosterol, 7‐dehydrocholesterol, and lathosterol were not detected, likely due to their extremely low abundance. As shown in Table S4, the ratios of these precursors to cholesterol were already very low in liver and plasma samples (< 0.005), and the total cholesterol content in bone tissues was substantially low, rendering quantification of these intermediates infeasible under the current analytical conditions. Further methodological refinement may be necessary to enable their quantification in these tissues. Additionally, phospholipid and triacylglycerol levels in bone marrow showed no significant differences (Figure 3c), indicating that the significant decrease in cholesterol and campesterol was not due to a reduction in fat content. The similar significant reduction in both cholesterol and campesterol levels in the bone marrow is considered to be due to their structural similarity.

FIGURE 3.

Lipids in the femur bone and marrow. (a) Sterol levels in the femur bone. (b) Sterol levels in the femur bone marrow. (c) Phospholipid and triacylglycerol levels in femoral bone marrow. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test.

3.4. The Variations in Plasma Cholesterol Were Opposite to Those Observed in the Cholesterol Levels in the in OVX Rats Bone Marrow

The sterols present in bone marrow tissue are supposed to be supplied from the blood. To further clarify the fluctuations of sterols in vivo, plasma cholesterol and campesterol levels were analyzed. As a result, plasma cholesterol levels were significantly increased by OVX treatment, while campesterol showed a tendency to increase (p = 0.0604). In contrast to their reduction in bone marrow, cholesterol and campesterol levels were elevated in plasma (Figure 4).

FIGURE 4.

The plasma squalene and sterol levels. (a) The cholesterol and precursors of cholesterol. (b) The phytosterols. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test.

3.5. No Significant Changes in Sterol Levels Were Observed in the Livers of OVX Rats

The liver, as a key organ in the synthesis and metabolism of cholesterol, regulates systemic cholesterol balance via its synthesis, transport, storage, and excretion. Dysregulated cholesterol metabolism can impair liver function and contribute to systemic diseases such as cardiovascular disorders. Therefore, hepatic sterol levels were also analyzed. In the analysis of liver sterols in the OVX and SHAM groups, both cholesterol and campesterol levels were reduced, but the differences were not statistically significant (Figure 5a,b).

FIGURE 5.

The liver squalene and sterol levels. (a) The cholesterol and precursors of cholesterol. (b) The phytosterols. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test.

3.6. Increased 4β‐Hydroxycholesterol‐To‐Cholesterol Ratio Suggests a Role in OVX‐Induced Osteoporosis

Cholesterol is oxidized to oxysterols through both automatic and enzymatic processes in vivo, and 4β‐hydroxycholesterol (4β‐OH) levels were significantly elevated in the humerus, plasma, and liver of the OVX group. On the other hand, in the bone marrow of the femur in the OVX group, several oxysterol levels were significantly decreased, while the levels of 4β‐hydroxycholesterol, 6‐ketocholesterol, and 27‐OH remained unchanged (Figure 6a). Given the significant reduction in cholesterol content in the OVX bone marrow, the oxysterol‐to‐cholesterol ratio was calculated. As a result, it was revealed that the ratio of 4β‐OH in the bone marrow was significantly increased in the OVX group (Figure 6b). These findings suggest a potential link between 4β‐OH and OVX‐induced osteoporosis.

FIGURE 6.

Oxysterol levels (a) and ratios to cholesterol (b) of the Humerus, Femur bone marrow, Plasma, and Liver. Data are presented as means ± S.E. (n = 5/group, *p < 0.05). Significant differences between mean values were evaluated by the Student's t‐test. OC: oxysterol; chol: cholesterol; 7α‐OH: 7α‐hydroxycholesterol; 7β‐OH: 7β‐hydroxycholesterol; β‐epoxy: 5β6β‐epoxycholesterol; 4β‐OH: 4β‐hydroxycholesterol; α‐epoxy: 5α6α‐epoxycholesterol; β‐triol: β‐cholestanetriol; 6‐keto: 6‐ketocholestanol; 25‐OH: 25‐hydroxycholesterol; 7‐keto: 7‐ketocholesterol; 27‐OH: 27‐hydroxycholesterol; 5α‐OH, 6‐keto: 5α‐hydroxy‐6‐keto‐cholesterol.

4. Discussion

This research was conducted using an OVX rat model of osteoporosis to investigate the content of various sterol molecules in the bone and bone marrow during the early stages of osteoporosis, with particular emphasis on the specific changes occurring in the bone and bone marrow. Following confirmation of successful OVX model induction, this study is the first to reveal the presence of sterol molecules in the bone and bone marrow. The results showed that the cholesterol and campesterol levels were significantly reduced in bone marrow during early osteoporosis, potentially contributing to reduced bone mineral density. However, the levels of cholesterol and campesterol in the bone remained unchanged. Furthermore, 4β‐OH was significantly elevated in the bone marrow. Therefore, cholesterol, campesterol, and 4β‐OH are suggested to be potential factors associated with the pathophysiology of OVX‐induced osteoporosis.

Among phytosterols, campesterol is not synthesized in vivo and has a low absorption rate (Bhattacharyya 1981). Previous reports have confirmed that dietary campesterol has been detected in the blood, muscles, liver, bile, and feces (Batta et al. 2006; Connor et al. 2005; Tomoyori et al. 2004). However, there has been no clear research on its involvement in bone tissue pathology. In this study, we confirmed the presence of campesterol in both bone and bone marrow. Structurally similar to cholesterol, campesterol may be incorporated into cell membranes (Grosjean et al. 2015; Begcevic Brkovic et al. 2023). In this study, in the OVX group, plasma cholesterol levels increased, while cholesterol content in the bone marrow decreased, and cholesterol content in the femur bone did not change. Campesterol followed a similar pattern to cholesterol. Therefore, both cholesterol and campesterol in the bone marrow were supplied from the blood, but no changes were observed in the cholesterol and campesterol content in the bone. This suggests that campesterol could serve as a marker to evaluate the uptake from blood into bone marrow. If campesterol is considered an indicator, bone cholesterol may be derived from bone marrow sterol pools, maintaining the balance of cholesterol. In the OVX group, no changes were observed in stigmasterol and β‐sitosterol in the bone. This is because the plasma level of β‐sitosterol remained unchanged. Additionally, stigmasterol and β‐sitosterol did not show a similar pattern to cholesterol compared to the campesterol since their lower absorption rates (Brufau et al. 2008) and structural differences, such as the presence of the ethyl group side chain difference in β‐sitosterol and stigmasterol instead of the methyl group in campesterol and the hydrogen atom in cholesterol at the C24 position or an additional double bond in stigmasterol at the C22–C23 position (Figure 7), may also influence phytosterol metabolic rates under the estrogen‐deficient condition. On the other hand, stigmasterol was not detected in the plasma, suggesting that it may accumulate in the bone and liver. In the bone of the femur, which structurally includes bone marrow, of the OVX group, while the cholesterol content increased, the campesterol content decreased. Furthermore, although the humerus, which has less bone marrow than the femur, tended to show an increase in cholesterol content, the levels of campesterol, β‐sitosterol remained similar. Thus, the observed bone density reduction in OVX rats appears unrelated to cholesterol levels in bone tissue in this study. It is also suggested that the supply of sterols from the bone marrow may influence the sterol content in the bone.

FIGURE 7.

The structures of cholesterol, campesterol, stigmasterol, and β‐sitosterol.

A significant increase in 4β‐OH was observed in the humerus, blood, and liver of the OVX group. On the other hand, in the femoral bone marrow of the OVX group, several oxysterol levels were significantly reduced, while the levels of 4β‐OH, 6‐ketocholesterol, and 27‐OH remained unchanged. However, when the oxysterol‐to‐cholesterol ratio was calculated, the ratio of 4β‐OH in the bone marrow of the OVX group had significantly increased. 4β‐OH is an oxidized derivative primarily generated in the liver (Mutemberezi et al. 2016) catalyzed by CYP3A4 and CYP3A5. The major enzyme of this reaction, CYP3A4, plays a crucial role in drug metabolism in the liver, and the levels of 4β‐OH are used as a biomarker of its activity (Mårde Arrhén et al. 2013; Karkhanis et al. 2024; Penzak and Rojas‐Fernandez 2019; Hole et al. 2018). Additionally, CYP3A4 and CYP3A5 have been reported to be expressed in mesenchymal stem cells and bone marrow macrophages in the bone marrow (Ghiaur et al. 2023; Chang et al. 2019; Alonso et al. 2015). Therefore, the increase in 4β‐OH in the femoral bone marrow of the OVX group may either originate from the liver via the bloodstream or be synthesized within the bone marrow itself. CYP3A4 and CYP3A5 are also involved in estrogen metabolism, producing hydroxylated estrogen metabolites with no estrogenic activity (Tsuchiya et al. 2005; Lee et al. 2003). Furthermore, 4β‐OH has been reported to activate LXR, thereby controlling cholesterol levels (Salonurmi et al. 2020; Nury et al. 2013). LXR is an important nuclear receptor involved in regulating cholesterol and lipid metabolism. The activation of LXR induces triacylglycerol accumulation (Bełtowski 2008; Ulven et al. 2005) and is also known to be involved in bone metabolism. Moreover, estrogen deficiency has been reported to cause bone marrow adiposity and a decrease in bone mass in both mice (Li et al. 2024) and humans (Li et al. 2020). These reports suggest that 4β‐OH may play a role in bone metabolism. In this study, since the triglyceride levels in the femoral bone marrow of the OVX group remained unchanged, indicating that the observed bone density reduction is likely independent of LXR‐mediated lipid accumulation. Furthermore, 4β‐OH has been shown to upregulate the expression of ABCG1 in macrophages, promoting cholesterol efflux (Lee and Tontonoz 2015). Osteoclasts, which are involved in bone metabolism, are differentiated from macrophages in the bone. Therefore, 4β‐OH may be involved in cholesterol metabolism in the bone marrow and affect the bone marrow microenvironment and bone metabolic pathways (such as the function of osteoblasts and osteoclasts), potentially contributing to the development of osteoporosis. Based on these prior studies, the function of 4β‐OH is not yet fully understood, but the increase in the ratio of 4β‐OH to cholesterol in the femoral bone marrow of the OVX group in this study is hypothesized to be associated with the decrease in bone density.

This study confirmed the presence of phytosterols, particularly campesterol, in the femur of the OVX‐induced osteoporosis model rats. Additionally, it was demonstrated that campesterol and cholesterol exhibited similar changes in the blood, liver, and bone marrow. Furthermore, 4β‐OH levels were significantly elevated in the bone marrow of OVX rats. These results suggest that 4β‐OH is involved in OVX‐induced osteoporosis and contributes to the decrease in bone density by influencing bone metabolism.

Author Contributions

W.H. and S.E. wrote the manuscript. W.H. and S.E. participated in the experiments and collected data. W.H., S.E., S.W., and Y.T. analyzed the data. W.H., S.E., S.W., Y.T., and M.S. designed the study. M.S. supervised the study and commented on the manuscript. All the authors have read and approved the final version of this manuscript.

Ethics Statement

This study was conducted with the approval of the Kyushu University Animal Experiment Committee (Approval No: A30‐313‐0).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Uterus weight and plasma cholesterol level (before selection).

Table S2: Major ion and confirmation ion of pre‐cholesterols, cholesterol and phytosterols in GC–MS analysis.

Table S3: Major ion and confirmation ion of oxysterols in GC–MS analysis.

Table S4: Precursors of cholesterol level ratios to cholesterol.

He, W. , Eguchi S., Wang S., Tanaka Y., and Sato M.. 2026. “Sterol Metabolism in Ovariectomized Rats: Potential Roles of Campesterol and 4β‐Hydroxycholesterol in Osteoporosis.” Lipids 61, no. 1: 151–162. 10.1002/lipd.70006.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.