Nested case-control study of one-carbon metabolites in mid-pregnancy and risks of cleft lip with and without cleft palate

Abstract

Evidence exists for an association between use of vitamin supplements with folic acid in early pregnancy and reduced risk for offspring with cleft lip with/without cleft palate (CLP). A few observations have been made about nutrients related to one-carbon metabolism other than folate. Our prospective study attempted to extend information on nutrition and CLP by measuring nutrient analytes in mid-pregnancy sera. This study included data from a repository of women’s mid-pregnancy serum specimens collected in California from 2003–04. Each woman’s specimen was linked with delivery information to determine whether her fetus had CLP or another structural malformation, or was nonmalformed. We identified 89 CLP cases. We randomly selected 409 specimens as controls. Specimens were tested for homocysteine, methylmalonic acid, folate, vitamin B₁₂, pyridoxal phosphate, pyridoxal, pyridoxic acid, riboflavin, choline, betaine, methionine, methionine sulfoxide, cysteine, cystathionine, arginine, and asymmetric and symmetric dimethylarginine. We observed three analytes with odds ratios unlikely to be explained by random variation, i.e., elevated CLP risks were observed for low levels and for high levels of pyridoxal phosphate (vitamin B₆), higher levels of choline, and low levels of symmetric dimethylarginine. These data did not show meaningful differences between cases and controls for any other analytes.

Evidence exists for an association between maternal use of a vitamin supplement with folic acid in early pregnancy and a reduced risk for offspring with cleft lip with/without cleft palate (CLP). This, evidence includes, i) epidemiologic studies that have shown reductions in both occurrence and recurrence (subsequent affected sibling) risks of CLP in infants whose mothers took vitamin supplements containing folic acid (1–11), ii) a reduction in prevalence of CLP following mandatory folic acid fortification in the US (12), iii) mothers’ use of folic acid antagonist medications associated with an increased risk to deliver offspring with CLP (13), and iv) biologic plausibility for a relation between folic acid and reduced risk for CLP from experimental studies (14).

Although folate has been the focus for many of the inquiries about periconceptional nutrition and clefting, there have been a few observations made about dietary intake or use of supplements involving nutrients related to one-carbon metabolism other than folate. We recently observed decreased risks of CLP associated with increasing intakes of total protein, alanine, choline, methionine, cysteine, iron and riboflavin (15). Investigators in the Netherlands have observed reduced risks of CLP associated with increased maternal dietary intakes of several B-vitamins, including thiamine, niacin, pyridoxine, and increased intakes of vegetable protein, fiber, beta-carotene, vitamin C, vitamin E, iron, and magnesium (16,17). Recent observations from the Danish prospective study found evidence to indicate that use of B₆ and B₁₂ supplements reduced risks of CLP (18).

A few studies have specifically measured nutrients in serum among women who delivered infants with CLP (19–22). These studies found serum concentrations that were lower in zinc (20), vitamin B₆ (19,21,22), vitamin B₁₂ (21), and higher in folate (22). However, these studies relied on post-partum measurements rather than measurements in early pregnancy, i.e., closer to the relevant embryonic timing for the development of the lip and palate which is gestational weeks 5–10. The current prospective study attempts to extend the knowledge base on nutrition and CLP by measuring numerous nutrient analytes in mid-pregnancy sera obtained from a sizable population in California.

Methods

This study included data from a large and unique mid-pregnancy serum specimen bank of pregnancies in California. Specimens were collected from approximately 70% of women during the 15^th-18^th week of pregnancy. These sera were collected from women who resided in selected regions of California (Orange, San Diego, and Central Valley counties). Sera were collected from women as part of the California Expanded AFP (alpha-feto-protein) program that screens for neural tube defects and cytogenetic abnormalities. The collection and processing of specimens was as follows: 1) samples were taken at draw stations using BD^™ Vacutainer 3.5 mL serum separator tubes with no anticoagulants or preservatives and centrifuged within 30 minutes; 2) samples were received by designated clinical laboratories from draw stations at room temperature, on average 3.0 days after draw; 3) AFP screening assays were run on samples usually on the day received; 4) samples were refrigerated up to 7 days if further testing was necessary; 5) samples were sent on cold packs via overnight mail to the serum storage bank; and 6) samples were aliquoted, labeled with barcodes, and frozen at −70°C within an average of 3.5 days of receipt at the serum storage bank.

Each woman’s serum specimen was linked with delivery outcome information to determine whether her fetus had CLP or any other structural malformation ascertained by the California Birth Defects Monitoring Program (23) or was born nonmalformed. The study included deliveries that were liveborn or stillborn (fetal deaths at greater than 20 weeks gestation) in 2003–2004. There were approximately 111,077 births represented in both the serum bank and the California Birth Defects Monitoring Program. Among these births, we identified 105 deliveries that involved CLP (cases). Of the 105, we excluded 16 with diagnoses involving holoprosencephaly, limb/body/wall defect, trisomy 13, or trisomy 18 owing to a suspected different underlying etiology. Of the 89 CLP cases available for further study, 80 were isolated, i.e., the phenotype did not involve any other major congenital abnormality. We also randomly selected 409 pregnancy specimens that were collected during the same time period and corresponded to nonmalformed infants at delivery (controls). Thus, this was a nested case-control study. All samples were obtained with approval from the California Health and Welfare Agency Committee for the Protection of Human Subjects.

Serum specimens for the 498 cases and controls were sent on cold packs to the University of Bergen for analyte measurements. The analytes measured were: total homocysteine, methylmalonic acid, folate, vitamin B₁₂, pyridoxal phosphate, pyridoxal, pyridoxic acid, riboflavin, total choline, betaine, methionine, methionine sulfoxide, total cysteine, cystathionine, asymmetric dimethylarginine, symmetric dimethylarginine, and creatinine. To assess cigarette smoking exposures, the metabolite cotinine was measured. No smoke exposure was defined as values <5 nmol/L and any smoke exposure was defined as ≥5 nmol/L. Details about all laboratory assays except total choline can be found elsewhere (24). Total choline was measured after conversion of choline esters to free choline in the presence of phospholipase D. A serum sample of 45 μL was mixed with 18 μL solution containing phospholipase D (Sigma Chemical Company, 2.8 U/μL), CaCl2 (86 mM), and Triton (0.44%). Then, 30 μL dithioerythritol (147mM) and 60 μL TCA containing 400 μM d7Choline were added to the incubation mixture. After centrifugation, the supernatant was analyzed by LC-MS/MS using a method optimized for the determination of free choline (24). Laboratory analyses were performed blind to case and control status.

We compared mean levels of analytes between cases and controls with t-tests. We also estimated risks using odds ratios and 95% confidence intervals (SAS 9.1). Models were constructed to assess effects associated with categories of the measured analytes. Specifically, we categorized measures as <25th percentile, 25th–74th percentile, and ≥75th percentile based on the distribution of each analyte among controls. The 25th-74th percentile was used as the reference group. We analyzed data for linear (logistic regression) and nonlinear (spline regression) effects and found no evidence for the latter. Available from intake forms associated with the screening program were the demographic factors maternal race/ethnicity (Hispanic; white, non Hispanic; Asian; Black; other) and maternal age (<25; 25–29; 30–34; and >34 years). These factors along with cigarette smoke exposure defined by cotinine levels were considered as covariates in some analyses.

Results

Table 1 shows similar racial/ethnic and age distributions between cases and controls. Table 2 shows means and standard deviations of each measured analyte. We observed statistically significantly (p≤0.05) lower mean levels in cases than in controls for vitamin B₁₂, betaine, methionine sulfoxide, total cysteine, and symmetric dimethylarginine. Higher mean levels of choline were observed among cases than controls. Removing 11 cases and 32 controls considered exposed to cigarette smoke based on cotinine levels did not substantially alter these findings (not shown).

Table 1.

Characteristics of cleft lip/palate-affected and unaffected (nonmalformed controls) deliveries, California 2003–05.

	Cleft Lip/ Palate Cases (n=89)		Controls (n=409)
	No.	%*	No.	%*
Race/Ethnicity
Hispanic	40	44.9	196	47.9
White nonHispanic	31	34.8	143	35.0
Asian	7	7.9	37	9.1
Black	2	2.3	12	2.9
Other	9	10.1	20	4.9
Age (years)
<25	29	32.6	132	32.3
25–29	26	29.2	91	22.3
30–34	24	27.0	137	33.5
≥34	10	11.2	49	12.0

^*Percentages may not equal 100 owing to missing data or rounding.

Table 2.

Mean values of selected biochemical measures in mid-pregnancy serum specimens among deliveries with cleft lip/palate and deliveries without malformations, California 2003–05.

	Cleft Lip/Palate Cases (n=89)		Controls (n=409)
Serum Measurements, mean (SD)	Mean	SD	Mean	SD	P-value
Homocysteine (tHcy) (μmol/L)	6.70	2.90	6.96	3.75	0.47
Methylmalonic acid (μmol/L)	0.16	0.09	0.15	0.05	0.28
Folate (nmol/L)	38.18	21.87	36.48	20.88	0.49
Vitamin B12 (pmol/L)	292.11	101.12	323.47	165.23	0.02
Pyridoxal phosphate (nmol/L)	67.41	52.39	72.19	63.17	0.45
Pyridoxal (nmol/L)	60.74	83.26	63.19	143.87	0.83
Pyridoxic acid (nmol/L)	61.56	88.26	64.47	153.27	0.81
Riboflavin (nmol/L)	35.58	26.17	39.88	29.86	0.21
Total choline (mmol/L)	3.09	0.46	2.98	0.41	0.02
Betaine (μmol/L)	15.82	3.66	16.69	4.64	0.05
Methionine (μmol/L)	36.92	10.58	36.28	9.72	0.58
Methionine sulfoxide (μmol/L)	1.68	0.73	1.99	1.37	0.003
Total cysteine (tCys) (μmol/L)	224.48	31.50	234.31	46.34	0.02
Cystathione (μmol/L)	0.13	0.07	0.12	0.05	0.07
Arginine (μmol/L)	159.67	41.24	151.22	50.85	0.10
Asymmetric dimethylarginine (μmol/L)	0.54	0.14	0.55	0.16	0.51
Symmetric dimethylarginine (μmol/L)	0.37	0.08	0.42	0.10	0.0001
Creatinine (μmol/L)	49.16	11.34	49.61	8.44	0.72

Given that comparisons of mean analyte values may not adequately reveal effects that could be associated with values toward the tails of the distribution, we explored lower and upper quartiles (cutpoints defined by the control distribution). As shown in Table 3, relative to the 25^th-75^th percentile, we observed three analytes (pyridoxal phosphate, choline, and symmetric dimethylarginine) that had associated odds ratios unlikely to be explained by random variation.

Table 3.

Effect estimates (odds ratios) for pregnancies affected with cleft lip/palate and selected biochemical measures in mid-pregnancy serum specimens.

Percentile Measure*	No. Cases (n=89)	No. Controls (n=409)	Odds Ratio	95% CI	Adjusted Odds Ratio**	95% CI
Total Homocysteine
< 4.90	22	101	1.1	0.6–2.0	1.1	0.6–2.0
4.90–7.14	40	205	Ref		Ref
≥7.15	27	103	1.3	0.8–2.3	1.4	0.8–2.4
p for trend		0.54			0.48
Methylmalonic acid
<0.12	17	98	0.7	0.4–1.3	0.7	0.4–1.3
0.12–0.16	50	206	Ref		Ref
≥0.17	22	105	0.9	0.5–1.5	0.9	0.5–1.5
p for trend		0.63			0.57
Folate, serum
<23.32	16	102	0.6	0.3–1.1	0.6	0.3–1.1
23.32–43.60	52	204	Ref		Ref
≥43.61	21	103	0.8	0.5–1.4	0.8	0.5–1.4
p for trend		0.51			0.47
Vitamin B12
<223.80	25	101	1.0	0.6–1.8	1.1	0.6–1.8
223.80–381.39	48	203	Ref		Ref
≥381.40	16	102	0.7	0.4–1.2	0.7	0.4–1.3
p for trend		0.21			0.23
Pyridoxal phosphate
<35.25	29	102	1.8	1.0–3.1	1.8	1.0–3.1
35.25–79.88	33	204	Ref		Ref
≥79.89	27	103	1.6	0.9–2.8	1.6	0.9–2.9
p for trend		0.77			0.72
Pyridoxal
<18.51	18	102	0.8	0.4–1.4	0.8	0.4–1.4
18.51–51.96	48	204	Ref		Ref
≥51.97	23	103	0.9	0.5–1.6	0.9	0.5–1.7
p for trend		0.51			0.53
Pyridoxic acid
<15.70	15	102	0.6	0.3–1.1	0.6	0.3–1.1
15.70–62.40	52	204	Ref		Ref
≥62.41	22	103	0.8	0.5–1.5	0.8	0.5–1.4
p for trend		0.35			0.36
Riboflavin
<22.34	28	102	1.2	0.7–2.1	1.2	0.7–2.1
22.34–46.65	45	204	Ref		Ref
≥46.66	16	103	0.7	0.4–1.3	0.7	0.4–1.3
p for trend		0.0979			0.0875
Choline, total
<2.71	17	100	1.0	0.5–1.8	1.0	0.5–1.8
2.71–3.20	36	206	Ref		Ref
≥3.21	36	102	2.0	1.2–3.4	2.1	1.2–3.5
p for trend		0.0133			0.0108
Betaine
<13.55	26	99	1.1	0.7–1.9	1.2	0.7–2.0
13.55–18.79	48	206	Ref		Ref
≥18.80	15	104	0.6	0.3–1.2	0.6	0.3–1.2
p for trend		0.0981			0.0900
Methionine
<29.41	20	102	0.8	0.5–1.5	0.9	0.5–1.5
29.41–41.32	48	204	Ref		Ref
≥41.33	21	103	0.9	0.5–1.5	0.9	0.5–1.6
p for trend		0.91			0.92
Methionine sulfoxide
<1.15	20	101	0.8	0.4–1.4	0.8	0.4–1.3
1.15–2.31	53	205	Ref		Ref
≥2.32	16	103	0.6	0.3–1.1	0.6	0.3–1.1
p for trend		0.54			0.54
Total cysteine
<207.50	24	102	1.0	0.6–1.7	0.9	0.5–1.6
207.50–245.59	50	204	Ref		Ref
≥245.60	15	103	0.6	0.3–1.1	0.6	0.3–1.1
p for trend		0.21			0.24
Cystathione
<0.08	11	85	0.6	0.3–1.2	0.6	0.3–1.1
0.08–0.12	47	209	Ref		Ref
≥0.13	31	115	1.2	0.7–2.0	1.2	0.7–2.0
p for trend		0.0617			0.0578
Arginine
<125.37	16	102	0.6	0.3–1.1	0.6	0.3–1.1
125.37–183.68	52	202	Ref		Ref
≥183.69	21	105	0.8	0.4–1.4	0.8	0.5–1.4
p for trend		0.55			0.48
Asymmetric dimethylarginine
<0.45	17	94	0.8	0.4–1.4	0.8	0.4–1.5
0.45–0.60	49	211	Ref		Ref
≥0.61	23	104	1.0	0.6–1.6	1.0	0.5–1.7
p for trend		0.59			0.63
Symmetric dimethylarginine
<0.34	33	92	1.6	1.0–2.7	1.6	0.9–2.6
0.34–0.47	48	214	Ref		Ref
≥0.48	8	103	0.3	0.2–0.8	0.3	0.2–0.7
p for trend		0.0002			0.0002
Asymmetric dimethylarginine/ Arginine*1000
<2.62	16	102	0.5	0.3–1.0	0.6	0.3–1.0
2.62–4.47	60	204	Ref		Ref
≥4.48	13	103	0.4	0.2–0.8	0.4	0.2–0.8
p for trend		0.65			0.57
Symmetric dimethylarginine/ Arginine*1000
<2.06	32	102	1.3	0.8–2.1	1.3	0.8–2.1
2.06–3.53	51	204	Ref		Ref
≥3.54	6	103	0.2	0.1–0.6	0.2	0.1–0.6
p for trend		0.0004			0.0003

^*Categories were constructed corresponding to <25, 25–74, and ≥75 percentiles, based on distributions among control mothers.

^**Odds ratio adjusted for maternal race/ethnicity and age.

For pyridoxal phosphate, elevated CLP risks were observed for low levels and for high levels. For choline, increased CLP risks were observed for higher levels. For symmetric dimethylarginine, increased CLP risks for low levels and decreased risks for high levels were observed. The risk pattern observed with choline and symmetric dimethylarginine each had statistically significant trend effects (Table 3). Adjustment of these analyses by maternal age and race/ethnicity (as well as folate, not shown) did not produce results that differed substantially from their unadjusted counterparts (Table 3). Analyses that excluded women with cigarette smoking exposure (11 case and 32 control women) also did not produce substantially different results.

We explored ratios of asymmetric dimethylarginine/arginine and symmetric dimethylarginine/arginine. These analyses revealed a highly significant trend for the symmetric dimethylarginine/arginine ratio (Table 3) with increasing ratio values associated with decreased CLP risk. Higher ratio values of asymmetric dimethylarginine/arginine were also associated with decreased CLP risk.

We simultaneously investigated four B-vitamin-related analytes, i.e., pyridoxal phosphate, riboflavin, betaine, and vitamin B₁₂. Compared to women whose levels were not in the lowest quartile for any of these four measures, women whose levels included at least one low quartile measure showed an odds ratio of 2.0 (1.1–3.5) adjusted for maternal age and race/ethnicity. Women with two or more low quartile measures showed similar effects (odds ratio=1.8 [1.0–3.2]) (data not shown).

Discussion

To our knowledge, this is the first prospective study conducted to examine potential associations between several serum nutrients related to one-carbon metabolism and CLP risk.

These data did not show a difference in mid-pregnancy serum folate levels between women whose pregnancies involved CLP and those whose pregnancies did not involve a structural malformation. The lack of an association with serum folate might be expected given that women were from a population whose food supply was fortified with folic acid and that most women likely were users of prenatal supplements containing folic acid as well as some of the other measured nutrients at the time of serum sampling. Our study shows notable findings for pyridoxal phosphate, choline, and symmetric dimethylarginine.

Pyridoxal phosphate is the predominant form of B₆ in human plasma (25). Similar to our observations, lower levels of this vitamin have been associated with an increased CLP risk in several populations (19,21,22,26), and there is experimental evidence to support an association between clefting and B₆. For example, mice fed a vitamin B₆-containing diet show a reduction in corticosteroid-induced clefting (27). In addition, our analyses involving a B-vitamin-related index (pyridoxal phosphate, riboflavin, betaine, and vitamin B₁₂) indicated low levels were associated with increased CLP risk. However, we also observed increased risks associated with higher levels, a finding not previously observed. Previous studies have collected specimens after delivery, often several years or more from the embryonic time of interest. The extent to which this design difference between current and previous work may contribute to differences in results is not clear. Pyridoxal phosphate has been shown to be stable for periods much longer and for freeze conditions at much higher temperatures than those associated with this study (25). However, our findings may be spurious given that pyridoxal phosphate is light sensitive (28) and we can not be sure that specimen handling avoided exposure to light. Further, some of our observations with pyridoxal phosphate were consistent with an interpretation of random fluctuation.

Choline, known primarily in the diet as a component of lecithin, is key to several metabolic processes. Like folate, choline is involved in one-carbon metabolism, it is utilized for the synthesis of cell membrane phospholipids, and it is a precursor of the neurotransmitter, acetylcholine (29–33). The demand for choline is thought to be higher during pregnancy (33). Our observed association with choline is unlikely to be explained by differential use of prenatal vitamin supplements between case and control mothers because choline is not a typical component of multivitamin supplements. However, the increased CLP risk observed for increased levels of choline is counter to our a priori expectation. Previous work by us showed higher dietary intakes of choline to be associated with decreased risk of CLP (15) and neural tube defects (34). We have also observed higher serum choline levels to be associated with decreased risks of neural tube defects (unpublished observations). Neural tube defects and CLP are believed to share embryologic features primarily based on a shared embryologic origin of cells contributing to the neural tube and to craniofacial development. We cannot provide a meaningful explanation as to why the risk pattern would be opposite to what we might have predicted. We do know that other aspects surrounding one-carbon metabolism have been shown to differ between CLP and neural tube defects, e.g., in some studies, the association with the TT genotype of the MTHFR gene appears to be a modest risk factor for neural tube defects (35) and somewhat of a protective factor for CLP (36,37).

A biologic explanation to support our findings for symmetric dimethylarginine and asymmetric dimethylarginine, that is, primarily decreased risks with increased levels, is unknown. Symmetric dimethylarginine is an indicator of renal function, asymmetric dimethylarginine inhibits nitric oxide synthase, and both are considered markers or risk factors for cardiovascular disease (38–40). We are not aware of any previous studies involving these analytes relative to risks of birth defects. Elevated asymmetric dimethylarginine has been negatively implicated in other reproductive outcomes such as pre-eclampsia and intrauterine growth retardation (41). Therefore, the relative importance of these analytes to CLP risk will need to be replicated in future work.

Limitations of this study include, first, the collection of women’s specimens on average 8 weeks after closure of lip and palate. It is likely that bias due to this single point-in-time measurement would tend to result in an underestimate of measured effects. A second limitation is potential degradation of analytes based on collection and storage procedures. It has been demonstrated that folate may degrade when frozen at higher temperatures (-20C) than were used for samples in this study (−80C) (42). Such degradation would likely be non-differential to case and control status and therefore tend to lead to underestimates of measured effects. Moreover, the average length of time between collection and frozen storage was similar between cases and controls. However, we explored whether even small differences between cases and controls influenced observed effects with analytes. Analyses that incorporated length of time into models produced similar odds ratios. Other limitations included a lack of information on supplemental and dietary intake of the nutrients studied here, a relatively small sample size that reduced power in some comparisons, findings for pyridoxal phosphate, choline, and symmetric dimethylarginine that may have arisen as a result of multiple comparisons, and an inability to investigate allelic variants of genes involved in the biosynthesis of some of the studied nutrients, e.g., folate, choline, B₆, and B₁₂. Additional study of genetic variants coupled with analyte measures would be informative owing to the known or suspected function of selected one-carbon metabolism- related genes and CLP (36,37,43–46).

Evidence continues to accumulate to show that nutrients, particularly folate, influence risks of structural birth defects. Our results substantially extend observations that vitamin B₆ may be important in craniofacial development. Our observations with choline also highlight the complexity of one-carbon metabolism as it relates to embryonic development.

Abbreviations

AFP

alpha-feto-protein

CLP

cleft lip with/without cleft palate