Determination of vitamin D status in singleton and twin gestations using CLIA and LC-MS/MS

in Endocrine Connections
Authors:
Magdalena Zgliczyńska Department of Obstetrics, Perinatology and Neonatology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Magdalena Ostrowska Department of Endocrinology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Kinga Żebrowska Department of Obstetrics, Perinatology and Neonatology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Iwona Szymusik Department of Obstetrics, Perinatology and Neonatology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Konrad Kowalski Masdiag Sp. z o.o., Stefana Żeromskiego, Warsaw, Poland

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Dorota Leszczyńska Department of Endocrinology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Katarzyna Kosińska-Kaczyńska Department of Obstetrics, Perinatology and Neonatology, Centre of Postgraduate Medical Education, Cegłowska, Warsaw, Poland

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Correspondence should be addressed to M Zgliczyńska: magdalena.zgliczynska@cmkp.edu.pl
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Objective

Vitamin D plays an important role during pregnancy. The aim was to compare vitamin D status in a group of singleton (SP) and twin pregnancies (TP) using two diagnostic methods: chemiluminescence immunoassay (CLIA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Design

This is a cross-sectional study.

Methods

The study was conducted in the population of SP and TP at the gestational age above 20 + 0 at the Bielanski Hospital in Warsaw, Poland, between October 2020 and January 2023. All patients had their venous blood samples collected and were given an original survey containing questions on demography and vitamin D supplementation.

Results

The study group included 53 Caucasian women with SP and 78 with TP aged from 21 to 47. Considering LC-MS/MS, patients with TP had lower concentrations of 25-hydroxyvitamin D (25(OH)D) than patients with SP. However, no significant difference was observed in the frequency of the occurrence of vitamin D deficiency (25(OH)D < 30 ng/mL). In both groups, the levels obtained with CLIA were significantly lower than in case of LC-MS/MS, however, strongly correlated. The intermethod agreement accounted for 52.4% and the Cohen’s kappa coefficient was 0.142.

Conclusions

The concentration of 25(OH)D in pregnant women depends on the type of gestation (SP/TP) and on the diagnostic methods used (CLIA/LC-MS/MS). Based on LC-MS/MS, the incidence of vitamin D deficiency was low in our group and no differences occurred in its frequency between SP and TP. The intermethod agreement between CLIA and LC-MS/MS on the detection of vitamin D deficiency was low.

Significance statement

This is the first study to compare the concentration of 25(OH)D levels between SP and TP using two methods: CLIA and the gold standard – LC-MS/MS. Based on LC-MS/MS, a low incidence of vitamin D deficiency was observed in our group, in which the vast majority of patients took cholecalciferol supplements. Moreover, there were no differences in its frequency between SP and TP. However, the 25(OH)D level was significantly lower in TP. The intermethod agreement between CLIA and LC-MS/MS on the detection of vitamin D deficiency was low, which is associated with substantial clinical implications.

Abstract

Objective

Vitamin D plays an important role during pregnancy. The aim was to compare vitamin D status in a group of singleton (SP) and twin pregnancies (TP) using two diagnostic methods: chemiluminescence immunoassay (CLIA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Design

This is a cross-sectional study.

Methods

The study was conducted in the population of SP and TP at the gestational age above 20 + 0 at the Bielanski Hospital in Warsaw, Poland, between October 2020 and January 2023. All patients had their venous blood samples collected and were given an original survey containing questions on demography and vitamin D supplementation.

Results

The study group included 53 Caucasian women with SP and 78 with TP aged from 21 to 47. Considering LC-MS/MS, patients with TP had lower concentrations of 25-hydroxyvitamin D (25(OH)D) than patients with SP. However, no significant difference was observed in the frequency of the occurrence of vitamin D deficiency (25(OH)D < 30 ng/mL). In both groups, the levels obtained with CLIA were significantly lower than in case of LC-MS/MS, however, strongly correlated. The intermethod agreement accounted for 52.4% and the Cohen’s kappa coefficient was 0.142.

Conclusions

The concentration of 25(OH)D in pregnant women depends on the type of gestation (SP/TP) and on the diagnostic methods used (CLIA/LC-MS/MS). Based on LC-MS/MS, the incidence of vitamin D deficiency was low in our group and no differences occurred in its frequency between SP and TP. The intermethod agreement between CLIA and LC-MS/MS on the detection of vitamin D deficiency was low.

Significance statement

This is the first study to compare the concentration of 25(OH)D levels between SP and TP using two methods: CLIA and the gold standard – LC-MS/MS. Based on LC-MS/MS, a low incidence of vitamin D deficiency was observed in our group, in which the vast majority of patients took cholecalciferol supplements. Moreover, there were no differences in its frequency between SP and TP. However, the 25(OH)D level was significantly lower in TP. The intermethod agreement between CLIA and LC-MS/MS on the detection of vitamin D deficiency was low, which is associated with substantial clinical implications.

Introduction

Vitamin D is a group of fat-soluble substances, traditionally classified as vitamins, but also acting as important hormones in the regulation of calcium and phosphate metabolism and bone mineralization in humans (1, 2, 3). In the natural habitat, it can be predominantly obtained by endogenous synthesis or, to a lesser extent, supplied through food (4). The process of the endogenous production of vitamin D in the human skin starts under the influence of UV B radiation, when 7-dehydrocholesterol is converted into previtamin D. Then, the vitamin D precursor derived from both skin and diet is hydroxylated in the liver to 25-hydroxyvitamin D (25(OH)D) and, in the next stage, to active 1,25-dihydroxyvitamin D (1,25(OH)2D), in the kidneys (5, 6). Vitamin D metabolites circulate in the blood bound particularly to vitamin D-binding protein (VDBP). As much as 10% is transported with albumins, whereas less than 0.1% remains a free fraction in healthy individuals (7, 8). After its release to tissues, 1,25(OH)2D acts on the vitamin D receptor (VDR), which is present in most tissues, and exerts numerous actions throughout the body (9).

The major changes in vitamin D metabolism during pregnancy are mostly associated with two main roles it plays during this special period: participation in providing the mother and the fetus with adequate calcium supply and the creation of maternal immune tolerance to the developing pregnancy (10, 11, 12). By the end of the first trimester, the concentration of 1,25(OH)2D in pregnant individuals usually increases, remaining high until delivery (10, 13). This is mainly due to an increase in its production in the kidney but also, to a lesser extent, in the placenta (14). In addition, the concentration of VDBP increases, thereby influencing the levels of free vitamin D metabolites. Lately, free metabolites have been proposed as potentially more representative of vitamin D status during pregnancy than total 25(OH)D, but the issue still remains unclear (11, 12, 13, 15). The fetus persists dependent on the placental transfer of maternal 25(OH)D (16). Cord blood concentrations of 25(OH)D account for 75–90% of those found in the maternal serum (17). However, despite the high popularity and the increasing prevalence of vitamin D supplementation, the problem of its deficiency in the pregnant population remains valid. In 2016, Saraf et al. systematically reviewed maternal and newborn vitamin D status and concluded that its deficiency, defined as 25(OH)D serum level below 20 ng/mL, might affect on average 54% of pregnant women and 75% of newborns worldwide, with high variability depending on the region (18). Women with multiple pregnancies (MP), the vast majority of which include twin pregnancies (TP), are a potentially vulnerable population. It was confirmed that they were associated with a higher metabolic demand; hence, it may be assumed that they might be linked with an increased need for individual nutrients (19, 20). One of the latest studies on vitamin D status in TP conducted in China showed a deficiency (<20 ng/mL) rate of 19.4% and insufficiency (20–30 ng/mL) rate of 20.8%, with 99.3% of neonates being deficient or insufficient, based on cord blood examination (21). Nevertheless, studies comparing vitamin D status between singleton pregnancies (SP) and MP published to date have shown conflicting results (19, 22, 23, 24, 25, 26, 27).

Until now, the measurement of 25(OH)D remains the principal way to determine vitamin D status in the general population mostly because it circulates in the blood in the highest concentration and has a long half-life (28). As for the methodology, competitive binding methods such as radioimmunoassays (RIA) or chemiluminescence immunoassays (CLIA) are predominantly used to assess 25(OH)D levels mostly due to their performance speed and cost-effectiveness. Nevertheless, an increasing quantity of data indicates a potentially large bias associated with those methods (28). Besides the method’s error itself, differences between the assays constitute an additional problem, which impedes the collective analysis of data obtained from different studies (28, 29). Liquid chromatography with tandem mass spectrometry (LC-MS/MS), which is free from numerous limitations of CLIA and other immunoassays, remains the gold standard for 25(OH)D testing (28). It also offers an additional benefit, i.e. the possibility of determining other vitamin D metabolites (30). Conversely, it is less available, more expensive, and time-consuming (28).

Aim of the study

The aim of the study was to compare vitamin D status in the group of SP and TP using two methods: CLIA and LC-MS/MS.

Materials and methods

General information

The STROBE (Strengthening the Reporting of Observational studies in Epidemiology) Guidelines were followed for reporting the study (5). This study is part of a larger project aimed at analyzing selected indicators of the nutritional status of pregnant women in TP in terms of key vitamins and trace elements which has not been completed and published at the time of writing this article. The subjects were recruited in the period between October 2020 and January 2023. We have first enrolled patients in TP counseled at the Endocrinology and Gynecology and Obstetrics Departments at Bielanski Hospital in Warsaw and then age-, gestational age-, and season-matched controls (SP) from the same site. A detailed description of the recruitment process is included in Fig. 1. Appropriate approval was obtained from the local ethics committee at the Centre of Postgraduate Medical Education (reference number 46/PB/2020 and 85/PB/2020), and all included patients gave their informed and written consent. All the patients received an original survey containing demographic questions as well as data on vitamin D supplementation before (at least for 1 month before obtaining a positive pregnancy test) and during pregnancy.

Figure 1
Figure 1

Recruitment process flowchart.

Citation: Endocrine Connections 12, 11; 10.1530/EC-23-0201

The inclusion criteria were as follows: the ability to make independent legal decisions about oneself, live pregnancy – twin or single, respectively, and gestational age calculated based on the last menstrual period and verified by the crown–rump length in the first trimester of pregnancy above 20 + 0 weeks. The exclusion criterion was the inability to give informed consent and known disorders affecting calcium and phosphate, as well as vitamin D metabolism (such as hyperparathyroidism, rickets, and sarcoidosis).

The context of the study and definitions used

Since it is considered that in Poland the synthesis of vitamin D in the skin may be sufficient in the period from May to September, we defined two periods for the purposes of the study: effective skin synthesis (from May to September) and ineffective skin synthesis (from October to April) (31). A serum level of total 25(OH)D 30–50 ng/mL was assumed to be the optimal supply of vitamin D (31).

The Polish Society of Obstetricians and Gynecologists recommendations endorse the supplementation of 1500–2000 IU of vitamin D in pregnant women irrespective of the number of fetuses. However, if possible, supplementation under the control of 25(OH)D concentration in the blood serum is favored (32).

Sample collection

Blood samples were collected at a single time point via venipuncture, after a 12-h overnight fasting, into 6.0 mL Vacuette tubes with a serum clot activator. The tubes were left at room temperature for 30 min to allow clotting, then centrifuged at 2000 g for 10 min. The serum samples were aliquoted and stored at −20°C.

Determination of 25(OH)D using CLIA

CLIA determination of 25(OH)D was performed with LIAISON® XL platform (DiaSorin, Italy) using direct competitive immunoassay LIAISON® 25(OH) Vitamin D TOTAL Assay. The first step involved incubation, during which 25(OH)D was dissociated from the VDBP and bound to the goat antibody on the solid phase. After 10 min, it was followed by adding vitamin D linked to an isoluminol derivative, which acts as a tracer. In this step, 25(OH)D in the test sample competes with the tracer for antibody binding sites. After 10 min of further incubation, unbound material was removed from the reaction vessel through a wash cycle. Then, starter reagents that initiate a flash chemiluminescence reaction are added. The light signal is measured using a photoluminometer and expressed as relative light units. The intra-assay coefficient of variation (CV) is 3.7–7.7%; the interassay CV is 4.8–7.7%. It shows 6.7% cross-reactivity with 1,25(OH)D2, 9.3% with 1,25(OH)D3 and minimal cross-reactivity with vitamin D3 (1.9%), vitamin D2 (1.9%) and 3-epi-25(OH)D3 (1.3%); the level of detection is 4.0 ng/ml.

Determination of 25(OH)D and other vitamin D metabolites using LC-MS/MS

The isotope dilution LC-MS/MS was used to analyze vitamin D metabolites (24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3, 3-epi 25(OH)D3). A two-stage protein precipitation protocol followed by derivatization was used for serum sample preparation. In the first step, 100 μL of the 25 ng/mL acetonitrile mixture of isotope-labeled vitamin D metabolites standards (13C5-25(OH)D3, 2H3-25(OH)D2, 2H6-24,25(OH)2D3) was added to 50 μL of the serum into a deep-96-well plate. After 10 min of equilibration on a horizontal shaker (60G, RT), the second portion of 300 μL of acetonitrile (IS) was added. The main precipitation was conducted for 30 min (60 g, RT) followed by centrifugation (10 min, 800 g, RT). In the next step, the supernatant (100 μL) was transferred to another polypropylene 96-well plate and evaporated under nitrogen stream to dryness (50°C). The samples then underwent derivatization using 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The dried residue was reconstituted in 100 μL of 200 mg/L DAPTAD solution in ethyl acetate and incubated in RT for 30 min in the absence of light. Specific reaction was stopped by the addition of 30 μL of methanol. The solution was again evaporated under nitrogen stream to dryness (50℃) and reconstituted in 100 μL of water:methanol mixture (1:1, v:v). Thus, the prepared samples were subjected (20 μL) to LC-MS/MS analysis. It was conducted using an ExionLC analytical high-performance liquid chromatography (HPLC) system with CTC PAL (Zwinger, Switzerland) autosampler coupled to the QTRAP® 5500 MS/MS system (Sciex, Framingham, MA, USA). All analyses were carried out in a positive mode using electrospray ionization. For quantitative analysis, multiple reaction monitoring was used. Chromatographic analyses were performed using Eclipse XDB-C18 1.7 m (50 × 4.6 mm) with a titanium prefilter at a flow rate of 0.8 mL/min. The column oven temperature was 40°C. The mobile phase included water (A) and acetonitrile (B), both with the addition of 0.1% formic acid. The gradient elution program was as follows: 0 min – 50% B, 2.5 min – 78% B, 3.2 min – 98% B, 4.5 min – 98% B, 4.6 min – 50% B. The total run time was 5.5 min. To compare the CLIA and LC-MS/MS methods, 25(OH)D3 and 25(OH)D2 concentrations were combined into 25(OH)D.

Statistical analyses

The power analysis for the study was carried out using the G-power program (33). Assuming an effect size of 1.38 estimated based on own measurements, with the significance level defined as 0.05 and the size of the group of 131 women, the calculated power amounted to 0.999, assuming an average difference of 25% between the results obtained with both methods.

Basic statistical analyses were performed using of Microsoft Excel (Microsoft Corporation) with STATISTICA 13.3.721.0 software (TIBCO Software Inc., Palo Alto, CA, USA). The normality of the distribution of the variables was assessed using the Shapiro–Wilk test. Therefore, we used the following parametric statistical test: Student’s t-test for the comparison of two independent or dependent groups and nonparametric statistical tests: Mann–Whitney U test, the Wilcoxon signed-rank, and Kruskal–Wallis ANOVA and median tests. Moreover, correlation matrices, Spearman’s correlations, crosstabulation, and Fisher’s exact tests were applied. The level of statistical significance was set at P < 0.05.

Subsequent analyses were performed in the R language in the RStudio environment (R-Tools Technology Inc., Richmond Hill, ON, Canada) and XLSTAT Life Science 2023.1.4. software (Lumivero Inc., Denver, CO, USA). The Passing–Bablok (P-B) regression and Bland–Altman charts were used to define the comparison between the results and the range of agreement between the two compared methods. Moreover, univariate and multivariate models were applied. To diagnose the models, the normality of the distribution of residuals was assessed and scedasticity was evaluated using a graphical interpretation.

Results

General characteristics

Among those who were asked to participate in the study, three women with SP and one with TP did not consent. The final study group consisted of 53 Caucasian women with SP and 78 women with TP aged from 21 to 47 years, permanently residing in Poland. Table 1 summarizes the basic comparative characteristics of the studied groups.

Table 1

Basic characteristics of the studied groups. Data are presented as median (IQR) or as n (%).

Singleton pregnancy Twin pregnancy P
Total (n) 53 78
Maternal age (years) 33 (31; 37) 35 (32; 37) 0.19
Gestational age (weeks) 31.3 (26.3; 35.4) 30.5 (25.4; 35.9) 0.40
Educationa
 Primary 0 (0.0%) 1 (1.6% ) 1.00
 Secondary 9 (19.6%) 9 (14.8%) 0.60
 Vocational 1 (2.2%) 1 (1.6%) 1.00
 Higher 36 (78.2%) 50 (82.0%) 0.63
Gravidity 2 (1; 3) 2 (1; 2) 0.29
Parity 0.21
 Primiparity 24 (45.3%) 45 (57.7%)
 Multiparity 29 (54.7%) 33 (42.3%)
Type of conception 0.03
 Natural 49 (92.5%) 60 (76.9%)
 Assisted 4 (7.5%) 18 (23.1%)
Common pregnancy complications
 Hypertensive disorders of pregnancy 10 (18.9%) 12 (15.4%) 0.60
 Gestational diabetes mellitus 4 (7.5%) 15 (19.2%) 0.06
Common comorbidities
 Asthma 3 (5.7%) 3 (3.9%) 0.69
 Hashimoto thyroiditis 7 (13.2%) 10 (12.8%) 1.00
 Arterial hypertension 3 (5.7%) 2 (2.6%) 0.39
 Any known autoimmune disorderb 10 (18.9%) 14 (17.9%) 1.00
Prepregnancy BMI (kg/m2) 23.0 (21.1; 27.1) 22.7 (21.1; 25.0) 0.43
Skin vitamin D synthesis – period of the year 0.11
 Effective (May to September) 10 (18.9%) 26 (32.5%)
 Ineffective (October to April) 43 (81.1%) 54 (67.5%)
Current supplementation of cholecalciferolc 1.00
 Yes 48 (96.0%) 66 (95.7%)
 No 2 (4.0%) 3 (4.3%)
Form of supplementationc
 Multivitamin 40 (80.0%) 49 (71.0%) 0.29
 Cholecalciferol only 2 (4.0%) 5 (7.2%) 0.70
 Both 6 (12.0%) 12 (17.4%) 0.45
 None 2 (4.0%) 3 (4.3%) 1.00
Current cholecalciferol dosage (IU)c 2000 (2000; 2000) 2000 (2000; 2000) 0.66
Supplementation of cholecalciferol before pregnancyd 0.13
 Yes 31 (62.0%) 31 (47.0%)
 No 19 (38.0%) 35 (53.0%)
Dosage of prepregnancy cholecalciferol supplementation (IU)d 2000 (0; 2000) 0 (0; 2000) 0.18

aData available for 107 women, 78.2% (n = 61) of women with twin and 86.8% (n = 46) with singleton pregnancy; bHashimoto thyroiditis, Graves’ disease, psoriasis, vitiligo, ankylosing spondylitis, ulcerative colitis; cData available for 119 women, 88.5% (n = 69) of women with twin and 94.3% (n = 50) with singleton pregnancy; dData available for 116 women, 84.6% (n = 66) of women with twin and 94.3% (n = 50) with singleton pregnancy.

BMI, body mass index; IQR, interquartile range.

The results of serum 25(OH)D levels using the CLIA and LC-MS/MS methods are presented in Table 2. There were no significant differences in the concentration of 25(OH)D between women recruited in effective and ineffective skin synthesis period for both diagnostic methods (CLIA median 32.3 ng/mL (interquartile range (IQR) 23.3–39.5) vs median 29.2 ng/mL (IQR 22.2–33.5), P = 0.055; LC-MS/MS median 46.5 ng/mL (IQR 37.6–61.3) vs median 47.3 ng/mL (IQR 37.6–54.3), P = 0.49, respectively).

Table 2

Results of 25(OH)D determinations using CLIA and LC-MS/MS.

Study group (n = 131) Singleton pregnancy (n = 53) Twin pregnancy (n = 78) P
25(OH)D by CLIA (ng/mL): median (IQR) 29.6 (22.2; 34.6) 27.2 (20.3; 31.7) 30.2 (24.8; 37.4) 0.02
25(OH)D by LC-MS/MS (ng/mL): median (IQR) 47.1 (37.6; 54.7) 49.2 (42.2; 57.7) 44.0 (36.0; 51.5) 0.01
P 0.00 0.00 0.00 -
Spearman’s correlation coefficient 0.60 0.66 0.69 -

25(OH)D, 25-hydroxyvitamin D; CLIA, chemiluminescence immunoassay; IQR, interquartile range; LC-MS/MS, liquid chromatography with tandem mass spectrometry.

Considering the CLIA method, patients with TP had statistically higher concentrations of 25(OH)D than patients with SP. However, regarding the results of LC-MS/MS, which is considered the reference method, patients with TP had statistically lower concentrations than patients with SP. However, no statistical difference was noted in the occurrence of vitamin D deficiency (defined as 25(OH)D < 30 ng/mL; TP – 9/78, 11.5%; SP – 4/53, 7.5%; P = 0.56). In both groups, the levels obtained with CLIA method were significantly lower than those of LC-MS/MS. However, they were strongly correlated.

Considering LC-MS/MS, both the concentration of 25(OH)D3 and 25(OH)D2 was significantly higher in the SP group, while the concentration of 3-epi 25(OH)D3 was lower than in the TP group. We have found no significant difference in the 24,25(OH)2D3 metabolite levels (Supplementary File 1, see section on supplementary materials given at the end of this article).

To compare and assess the equivalence of CLIA and LC-MS/MS results, P-B regression was used (y = 0.6783x – 1.5038). Considering both a = 0 and b = 1 are true at 95% confidence intervals (CI 95%) (intercept: −6461–2826 ng/mL, slope: 0.573–0.789), it can be concluded that the two methods are not interchangeable and there is a proportional bias between methods (Fig. 2).

Figure 2
Figure 2

Passing–Bablok correlation between concentrations of 25(OH)D obtained with LC-MS/MS and CLIA methods.

Citation: Endocrine Connections 12, 11; 10.1530/EC-23-0201

The Bland–Altman analysis confirmed a substantial difference between the result obtained using LC-MS/MS and CLIA methods (Fig. 3A). CLIA 25(OH)D concentration was on average lower for 19 ng/mL (CI 95%: −37 to 3 ng/mL) compared to LC-MS/MS measurement. Simultaneously, a trend was identified where the difference between the results from the two methods increased in accordance with the increasing concentration of 25(OH)D. To confirm this observation relative Bland–Altman plot was also performed (Fig. 3B). Obtained results showed 49.3% average bias between two methods with CI 95% between −96.8% and 7.36% with total seven residuals exceeding limits of agreement (9.2% of observations). That analysis confirmed a strong proportional bias between both methods.

Figure 3
Figure 3

(A) Bland–Altman plot of differences in 25(OH)D concentration measured with CLIA and LC-MS/MS methods. (B) Relative Bland–Altman plot of differences in 25(OH)D concentration measured with CLIA and LC-MS/MS methods.

Citation: Endocrine Connections 12, 11; 10.1530/EC-23-0201

The results of linear modeling allowed the derivation of a formula for calculating the expected level of 25(OH)D in the LC-MS/MS method based on the known result in CLIA. The coefficient of determination was 0.41, which in the context of comparing those two methods is a relatively low result (Fig. 4 and Table 3).

Figure 4
Figure 4

One-factor linear regression model for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS.

Citation: Endocrine Connections 12, 11; 10.1530/EC-23-0201

Table 3

One-factor linear regression model for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS.

Beta Standard error T P
Intercept 21.05154 2.81423 7.48 <0.01
CLIA 0.86049 0.08998 9.563 <0.01
R2 = 0.4148

CLIA, chemiluminescence immunoassay.

The two-factor model concerning the occurrence of TP showed a significant dependence of the level of 25(OH)D on the type of pregnancy. In mothers with SP, the level of 25(OH)D determined by the reference LC-MS/MS method, was on average 9.6 ng/mL higher than in women with TP. After adjusting for the type of pregnancy, the model coefficient of determination increased to 0.52 (Fig. 5 and Table 4). For both models, the average residual was 0 with the normal distribution of residuals. In both cases, the models were homoscedastic.

Figure 5
Figure 5

Multivariate regression model for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS with correction for multiple pregnancies.

Citation: Endocrine Connections 12, 11; 10.1530/EC-23-0201

Table 4

Linear model comprising the impact of multiple pregnancies for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS.

Beta Standard error T P
Intercept 24.0274 2.5951 9.259 <0.01
CLIA 0.9536 0.0829 11.502 <0.01
Twin pregnancy −9.6139 1.7399 −5.525 <0.01
R 2 = 0.5201

CLIA , chemiluminescence immunoassay.

In the next step, patients suffering from vitamin D deficiency were identified based on both measurement methods, and the results were subjected to a compatibility analysis (Table 5). The intermethod agreement was 52.4%, and Cohen’s kappa coefficient was 0.142 (CI 95% 0.05–0.23). In the analysis of the compatibility of vitamin D deficiency diagnoses, a compliance rate of 52% was observed.

Table 5

Compliance analysis of vitamin D deficiency diagnosed assuming a threshold of 30 ng/mL.

CLIA LC-MS/MS Total
No deficiency % Deficiency %
No deficiency 59 45.04% 1 0.76% 60
Deficiency 59 45.04% 12 9.16% 71
Total 118 13

CLIA, chemiluminescence immunoassay; LC-MS/MS, liquid chromatography with tandem mass spectrometry.

By performing additional analyses, we have also found a correlation between the dosage of current cholecalciferol supplementation and the levels of 25(OH)D obtained by LC-MS (R = 0.36; P < 0.05). Detailed results are collected in Supplementary File 2. We have also compared women taking only combined supplement (n = 89) with the group supplementing vitamin D in separate formula (n = 7). There was no significant difference in the supplemented dose between these groups (median 2000 IU (2000; 2000) vs median 2000 IU (2000; 2000); P = 0.23) and no differences were found in the level of 25(OH)D by CLIA (median 29.8 ng/mL (24.2; 34.7) vs median 29.4 ng/mL (15.4; 30.0); P = 0.38), and LC-MS (median 46.4 ng/mL (38.6; 53.2) vs 37.8 ng/mL (19.7; 56.1); P = 0.52).

In the next step, we compared patients with and without hypertensive disorders of pregnancy (HDP) as well as gestational diabetes mellitus (GDM) and found no significant differences in the level of 25(OH)D measured in LC-MS/MS (HDP median 48.5 ng/mL (IQR 43.0–54.2) vs no-HDP 46.9 ng/mL (IQR 37.6–54.7), P = 0.43; GDM 48.3 ng/mL (IQR 42.7-51.8) vs no-GDM 47.0 ng/mL (IQR 37.5–55.3), P = 0.96). We also found no significant differences in the level of 25(OH)D using CLIA and LC-MS between patients with no known autoimmunological disease and patients with concomitant autoimmunity (CLIA median 29.6 ng/mL (IQR 22.2–35.1) vs 29.6 ng/mL (IQR 25.4–32.9), P = 0.98; LC-MS/MS median 47.2 ng/mL (IQR 38.4–54.2) vs 46.7 ng/mL (IQR 35.3–55.5), P = 0.95 respectively). As gestational weight gain is a multifactorial variable, in order to unify the study groups in the next analysis we took into account only women in the third trimester of pregnancy and divided them into two categories: excessive weight gain (n = 12) and others (n = 73) (in accordance with the Institute of Medicine guidelines from 1990, updated in 2009) (34). There were no significant differences in the 25(OH)D measured by LC-MS between women with excessive weight gain and the others (median 43.8 ng/mL (IQR 37.4–51.2) vs median 49.0 ng/mL (IQR 37.8–57.1); P = 0.23), but there were significant differences between the groups in the level of 25(OH)D measured by CLIA (median 22.0 ng/mL (IQR 19.4–27.9) vs 29.6 ng/mL (IQR 24.6–36.5); P = 0.004).

Discussion

In this study, by using the diagnostic method currently considered a gold standard in the assessment of vitamin D status, namely, LC-MS/MS, we noted lower levels of 25(OH)D in women with TP than those in women with SP, with no statistical difference in the occurrence of vitamin D deficiency (defined as 25(OH)D below 30 ng/mL) between the groups. However, few studies conducted so far on this group of patients have presented conflicting results. Reddy et al. and Okah et al. used HPLC. Similar to our results, the first mentioned group of authors showed lower concentrations of 25(OH)D in TP than in SP. However, the second group reported the concentrations to be higher (25, 26). Nakayama et al., who used the RIA method, showed lower levels of 25(OH)D in a group of women with TP, as did Goswami et al. and Blarduni et al., who determined them with the use of CLIA (22, 23, 24). So far, to our knowledge, only two such studies using the LC-MS/MS technology have been published. Le et al. reported higher concentrations of 25(OH)D in TP than in SP and also a significantly higher incidence of vitamin D deficiency (according to the Institute of Medicine <12 ng/mL) in SP (27). Li et al. did not perform a comparative analysis but showed an average 25(OH)D concentration of 31.8 ng/mL in TP. However, the dose supplemented by the patients was also lower than in our cohort (500 IU/day) and the participants were an ethnically different group (21). Therefore, another conclusion to be drawn from our study is that the problem of vitamin D deficiency, defined as the concentration of the 25(OH)D metabolite below 30 ng/mL, was not common in our population, and this was probably due to the high prevalence of supplementation during pregnancy, which influenced the level of 25(OH)D.

Another important aspect of the study is the demonstration of colossal differences in the results obtained with the two methods: CLIA and LC-MS/MS. Nevertheless, numerous studies published so far have also confirmed large disparities between the results of 25(OH)D determinations using various methods (35, 36). In fact, they seem so prominent that there are even attempts to create predictive models aimed at standardizing historical data from studies in which immunoassays were mainly used (37). Nevertheless, the positive correlation between the concentration of 25(OH)D in LC-MS/MS and the difference between LC-MS/MS and CLIA was our interesting and innovative observation. On this basis, we assumed that as the concentration of 25(OH)D increased, the discrepancy between the methods became greater, which might have resulted in the above-described divergent outcomes obtained with the two methods. Low reliability of immunoassays could be due to several reasons, which include cross-reactivity between vitamin D metabolites and poor specificity of the used antibodies (28). During pregnancy, these differences may be further enhanced by altered vitamin D metabolism. As mentioned earlier, pregnancy results in a higher concentration of VDBP. This may contribute to greater errors in concentration determination with immunoassays because they are based on the dissociation of 25(OH)D from its binding protein (28). The above-mentioned study by Le et al. revealed higher concentrations of VDBP in TP than in SP. The authors suggested that this may have contributed to higher 25(OH)D levels demonstrated in TP, emphasizing the simultaneous lack of differences in free 25(OH)D levels between TP and SP (27). Some studies also show that changes in VDBP levels might be partially correlated with body mass index (BMI), which could be one of the reasons for the different results obtained with CLIA than LC-MS/MS in women with excessive pregnancy weight gain and others (38, 39).

As it was shown in our study, such differences may have huge clinical implications. According to Polish recommendations, vitamin D supplementation during pregnancy should ideally be based on its serum concentration (31, 32). In this case, many women examined with the CLIA method undergo, as it often turns out, an unnecessary therapeutic intervention based on the results obtained with LC-MS/MS (28). This is especially important as some authors reported negative effects of high vitamin D levels in offspring. However, ‘excess’ has not been defined in this group yet. Vitamin D toxicity may occur in healthy adult individuals when 25(OH)D levels reach above 100 ng/mL, while the serum level at which fetal development and life may be impaired remains unknown (31, 40). For instance, Gale et al., based on their data, concluded that the maternal concentrations of 25(OH)D of over 30 ng/mL did not appear to influence the child’s intelligence, psychological health, or cardiovascular system, but there could be an increased risk of eczema and asthma (41). Therefore, the true desired level of all vitamin D metabolites, considering all current and long-term effects, effects on the bone, and the pleiotropic effects of vitamin D in pregnant women, especially with TP, and their neonates, is yet to be determined (42).

Strengths and limitations

The main advantage of this study is the fact that we have examined vitamin D status in a special group of patients: women with TP and compared the results to SP. In addition, we used two methods: LC-MS/MS, which is a reference method, and CLIA, which is one of the most frequently used methods to assess the 25(OH)D in the same blood sample (28). The groups were well matched with respect to ethnicity, maternal age, gestational age, prepregnancy BMI, seasons, and supplementation use. Nevertheless, there might be doubts about selection bias, since some of the potentially eligible patients were missed due to reasons described in Fig. 1. Moreover, the recruitment was carried out in the hospital, which may be conducive to sample selection bias, although in our opinion, in Poland, where the vast majority of patients receive physician care throughout the pregnancy and give birth in hospital, this should not be a source of significant distortion. The limitations of our study include its observational nature, nonunified supplementation dose, and the fact that the studies were performed at a single time point of pregnancy. As mentioned earlier, additional valuable information could be obtained by measuring other vitamin D metabolism-associated markers, such as VDBP or free vitamin D metabolites.

Conclusions

The concentration of 25(OH)D in pregnant women is dependent on the type of gestation: singleton or twin and on the determination methods used. Based on the reference method, which is LC-MS/MS, the incidence of vitamin D deficiency in our study group was low and there were no significant differences between SP and TP which was probably influenced by the high prevalence of supplementation. Results obtained with CLIA and LC-MS/MS were highly correlated, but regarding a considerable shift between results obtained with two methods, the intermethod agreement on the detection of vitamin D deficiency was low. Since it has a significant impact on clinical management, we conclude that the results obtained with CLIA need to be treated with caution in this cohort.

Supplementary materials

This is linked to the online version of the article at https://doi.org/10.1530/EC-23-0201.

Declaration of interest

All authors declare no conflict of interest.

Funding

This work was supported by the Centre of Postgraduate Medical Education, Warsaw, Poland – grant numbers 506-1-022-01-21, 506-1-022-01-22, and 506-1-157-01-23.

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  • Collapse
  • Expand
  • Figure 1

    Recruitment process flowchart.

  • Figure 2

    Passing–Bablok correlation between concentrations of 25(OH)D obtained with LC-MS/MS and CLIA methods.

  • Figure 3

    (A) Bland–Altman plot of differences in 25(OH)D concentration measured with CLIA and LC-MS/MS methods. (B) Relative Bland–Altman plot of differences in 25(OH)D concentration measured with CLIA and LC-MS/MS methods.

  • Figure 4

    One-factor linear regression model for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS.

  • Figure 5

    Multivariate regression model for the relationship between the measurements of the compared methods: CLIA and LC-MS/MS with correction for multiple pregnancies.

  • 1

    Pludowski P, Holick MF, Grant WB, Konstantynowicz J, Mascarenhas MR, Haq A, Povoroznyuk V, Balatska N, Barbosa AP, Karonova T, et al.Vitamin D supplementation guidelines. Journal of Steroid Biochemistry and Molecular Biology 2018 175 125135. (https://doi.org/10.1016/j.jsbmb.2017.01.021)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 2

    Chang SW, & Lee HC. Vitamin D and health - the missing vitamin in humans. Pediatrics and Neonatology 2019 60 237244. (https://doi.org/10.1016/j.pedneo.2019.04.007)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3

    Sassi F, Tamone C, & D'Amelio P. Vitamin D: nutrient, hormone, and immunomodulator. Nutrients 2018 10. (https://doi.org/10.3390/nu10111656)

  • 4

    Polzonetti V, Pucciarelli S, Vincenzetti S, & Polidori P. Dietary intake of vitamin D from dairy products reduces the risk of osteoporosis. Nutrients 2020 12. (https://doi.org/10.3390/nu12061743)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5

    von Elm E, Altman DG, Egger M, Pocock SJ, Gotzsche PC, Vandenbroucke JP & STROBE Initiative. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Journal of Clinical Epidemiology 2008 61 344349. (https://doi.org/10.1016/j.jclinepi.2007.11.008)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6

    Christakos S, Li S, De La Cruz J, & Bikle DD. New developments in our understanding of vitamin metabolism, action and treatment. Metabolism: Clinical and Experimental 2019 98 112120. (https://doi.org/10.1016/j.metabol.2019.06.010)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7

    Bikle DD, & Schwartz J. Vitamin D binding protein, total and free vitamin D levels in different physiological and pathophysiological conditions. Frontiers in Endocrinology (Lausanne) 2019 10 317. (https://doi.org/10.3389/fendo.2019.00317)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8

    Abdulrahman MA, Alkass SY, & Mohammed NI. Total and free vitamin D status among apparently healthy adults living in Duhok Governorate. Scientific Reports 2022 12 1778. (https://doi.org/10.1038/s41598-022-05775-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9

    Pike JW, Meyer MB, Lee SM, Onal M, & Benkusky NA. The vitamin D receptor: contemporary genomic approaches reveal new basic and translational insights. Journal of Clinical Investigation 2017 127 11461154. (https://doi.org/10.1172/JCI88887)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10

    Hollis BW, & Wagner CL. New insights into the vitamin D requirements during pregnancy. Bone Research 2017 5 17030. (https://doi.org/10.1038/boneres.2017.30)

  • 11

    Karras SN, Wagner CL, & Castracane VD. Understanding vitamin D metabolism in pregnancy: from physiology to pathophysiology and clinical outcomes. Metabolism: Clinical and Experimental 2018 86 112123. (https://doi.org/10.1016/j.metabol.2017.10.001)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12

    Fernando M, Ellery SJ, Marquina C, Lim S, Naderpoor N, & Mousa A. Vitamin D-binding protein in pregnancy and reproductive health. Nutrients 2020 12. (https://doi.org/10.3390/nu12051489)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13

    Best CM, Pressman EK, Queenan RA, Cooper E, & O'Brien KO. Longitudinal changes in serum vitamin D binding protein and free 25-hydroxyvitamin D in a multiracial cohort of pregnant adolescents. Journal of Steroid Biochemistry and Molecular Biology 2019 186 7988. (https://doi.org/10.1016/j.jsbmb.2018.09.019)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

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