Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19.

SARS-CoV-2, the causative agent of COVID-19 disease, has resulted in the death of millions worldwide since the beginning of the pandemic in December 2019. While much progress has been made to understand acute manifestations of SARS-CoV-2 infection, less is known about post-acute sequelae of COVID-19 (PASC). We investigated the levels of both Spike protein (Spike) and viral RNA circulating in patients hospitalized with acute COVID-19 and in patients with and without PASC. We found that Spike and viral RNA were more likely to be present in patients with PASC. Among these patients, 30% were positive for both Spike and viral RNA; whereas, none of the individuals without PASC were positive for both. The levels of Spike and/or viral RNA in the PASC+ve patients were found to be increased or remained the same as in the acute phase; whereas, in the PASC-ve group, these viral components decreased or were totally absent. Additionally, this is the first report to show that part of the circulating Spike is linked to extracellular vesicles without any presence of viral RNA in these vesicles. In conclusion, our findings suggest that Spike and/or viral RNA fragments persist in the recovered COVID-19 patients with PASC up to 1 year or longer after acute SARS-CoV-2 infection.

Altered dietary ratio of folic acid and vitamin B12 during pregnancy influences the expression of imprinted H19/IGF2 locus in C57BL/6 mice.

Maternal folic acid and vitamin B12 (B12) status during pregnancy influence fetal growth. This study elucidated the effect of altered dietary ratio of folic acid and B12 on the regulation of H19/IGF2 locus in C57BL/6 mice. Female mice were fed diets with nine combinations of folic acid and B12 for 4 weeks. They were mated and the offspring born (F1) were continued on the same diet for 6 weeks post-weaning and were allowed to mate. The placenta and fetal (F2) tissues were collected at day 20 of gestation. H19 overexpression observed under dietary deficiency of folate combined with normal B12 (B12 normal folic acid-deficient, BNFD) was associated with an increased expression of microRNA-675 (miR-675) in maternal and fetal tissues. Insulin-like growth factor 2 (IGF2) expression was decreased under folic acid-deficient conditions combined with normal, deficient or over-supplemented state of B12 (BNFD, BDFD and BOFD) in fetal tissues along with B12 deficiency combined with normal folic acid (BDFN) in the placenta. The altered expression of imprinted genes under folic acid-deficient conditions was related to decreased serum levels of folate and body weight (F1). Hypermethylation observed at the H19 differentially methylated region (DMR) (in BNFD) might be responsible for the decreased expression of IGF2 in female fetal tissues. IGF2 DMR2 was found to be hypomethylated and associated with low serum B12 levels with B12 deficiency in fetal tissues. Results suggest that the altered dietary ratio of folic acid and B12 affects the in utero development of the fetus in association with altered epigenetic regulation of H19/IGF2 locus.

Extracellular Vesicle TGF-ß1 Is Linked to Cardiopulmonary Dysfunction in Human Immunodeficiency Virus.

Extracellular vesicles (EVs) have emerged as important mediators in cell-cell communication; however, their relevance in pulmonary hypertension (PH) secondary to human immunodeficiency virus (HIV) infection is yet to be explored. Considering that circulating monocytes are the source of the increased number of perivascular macrophages surrounding the remodeled vessels in PH, this study aimed to identify the role of circulating small EVs and EVs released by HIV-infected human monocyte-derived macrophages in the development of PH. We report significantly higher numbers of plasma-derived EVs carrying higher levels of TGF-ß1 (transforming growth factor-ß1) in HIV-positive individuals with PH compared with individuals without PH. Importantly, levels of these TGF-ß1-loaded, plasma-derived EVs correlated with pulmonary arterial systolic pressures and CD4 counts but did not correlate with the Dl CO or viral load. Correspondingly, enhanced TGF-ß1-dependent pulmonary endothelial injury and smooth muscle hyperplasia were observed. HIV-1 infection of monocyte-derived macrophages in the presence of cocaine resulted in an increased number of TGF-ß1-high EVs, and intravenous injection of these EVs in rats led to increased right ventricle systolic pressure accompanied by myocardial injury and increased levels of serum ET-1 (endothelin-1), TNF-α, and cardiac troponin-I. Conversely, pretreatment of rats with TGF-ß receptor 1 inhibitor prevented these EV-mediated changes. Findings define the ability of macrophage-derived small EVs to cause pulmonary vascular modeling and PH via modulation of TGF-ß signaling and suggest clinical implications of circulating TGF-ß-high EVs as a potential biomarker of HIV-associated PH.

Different dietary combinations of folic acid and vitamin B12 in parental diet results in epigenetic reprogramming of IGF2R and KCNQ1OT1 in placenta and fetal tissues in mice.

Genomic imprinting is important for mammalian development and its dysregulation can cause various developmental defects and diseases. The study evaluated the effects of different dietary combinations of folic acid and B12 on epigenetic regulation of IGF2R and KCNQ1OT1 ncRNA in C57BL/6 mice model. Female mice were fed diets with nine combinations of folic acid and B12 for 4 weeks. They were mated and off-springs born (F1) were continued on the same diet for 6 weeks postweaning and were allowed to mate. The placenta and fetal (F2) tissues were collected at day 20 of gestation. Dietary deficiency of folate (BNFD and BOFD) and B12 (BDFN) with either state of other vitamin or combined deficiency of both vitamins (BDFD) in comparison to BNFN, were overall responsible for reduced expression of IGF2R in the placenta (F1) and the fetal liver (F2) whereas a combination of folate deficiency with different levels of B12 revealed sex-specific differences in kidney and brain. The alterations in the expression of IGF2R caused by folate-deficient conditions (BNFD and BOFD) and both deficient condition (BDFD) was found to be associated with an increase in suppressive histone modifications. Over-supplementation of either folate or B12 or both vitamins in comparison to BNFN, led to increase in expression of IGF2R and KCNQ1OT1 in the placenta and fetal tissues. The increase in the expression of IGF2R caused by folate over-supplementation (BNFO) was associated with decreased DNA methylation in fetal tissues. KCNQ1OT1 noncoding RNA (ncRNA), however, showed upregulation under deficient conditions of folate and B12 only in female fetal tissues which correlated well with hypomethylation observed under these conditions. An epigenetic reprograming of IGF2R and KCNQ1OT1 ncRNA in the offspring was evident upon different dietary combinations of folic acid and B12 in the mice.

Temporal expression of genes involved in folate metabolism and transport during placental development, preeclampsia and neural tube defects.

Folate is an essential micronutrient during pregnancy. The differential expression of genes related to folate transport and metabolism during the advancing gestation and pregnancy complications is not well established. Hence, we studied the gene expression of folate metabolism and transport proteins in the placenta with advancing gestation, preeclampsia and neural tube defects (NTD). The expression of folate transporters and enzymes involved in folate metabolism in the placenta with advancing gestation and pregnancy-related disorders were studied by 2-step RT-PCR. Folate levels were estimated by microbiological assay using Lactobacillus casei. Significant changes in levels of placental folate metabolizing enzymes were found in both physiological and pathological pregnancies during advancing gestation. Expression of methyltetrahydrofolate reductase (MTHFR) (p < 0.001) and cystathionine-ß-synthase (CBS) (p < 0.001) was decreased while that of methionine synthase (MS) (p < 0.001) was increased with advancing gestation. A much-reduced expression of MTHFR (p < 0.01) and an abnormally high expression of methionine synthase reductase (p < 0.001) were observed in the NTD group. In NTDs, there was an adaptive up-regulation of folate transporters mainly reduced folate carrier (p < 0.001) and folate receptor alpha (p < 0.001). MTHFR expression showed a strong positive correlation (r = 0.96, p < 0.01) with folate levels in placenta. Pregnant women with preeclampsia had low expression of MS (p < 0.01) in association with low folate levels. Placental folate metabolizing enzymes exhibited a differential pattern during advancing gestation. Deficient folate status in association with alteration in expression of enzymes involved in folate metabolism might be associated with pregnancy complications such as preeclampsia and NTDs.

Analysis of MicroRNA Cargo in Circulating Extracellular Vesicles from HIV-Infected Individuals with Pulmonary Hypertension.

The risk of developing pulmonary hypertension (PH) in people living with HIV is at least 300-fold higher than in the general population, and illicit drug use further potentiates the development of HIV-associated PH. The relevance of extracellular vesicles (EVs) containing both coding as well as non-coding RNAs in PH secondary to HIV infection and drug abuse is yet to be explored. We here compared the miRNA cargo of plasma-derived EVs from HIV-infected stimulant users with (HIV + Stimulants + PH) and without PH (HIV + Stimulants) using small RNA sequencing. The data were compared with 12 PH datasets available in the GEO database to identify potential candidate gene targets for differentially altered miRNAs using the following functional analysis tools: ingenuity pathway analysis (IPA), over-representation analysis (ORA), and gene set enrichment analysis (GSEA). MiRNAs involved in promoting cell proliferation and inhibition of intrinsic apoptotic signaling pathways were among the top upregulated miRNAs identified in EVs from the HIV + Stimulants + PH group compared to the HIV + Stimulants group. Alternatively, the downregulated miRNAs in the HIV + Stimulants + PH group suggested an association with the negative regulation of smooth muscle cell proliferation, IL-2 mediated signaling, and transmembrane receptor protein tyrosine kinase signaling pathways. The validation of significantly differentially expressed miRNAs in an independent set of HIV-infected (cocaine users and nondrug users) with and without PH confirmed the upregulation of miR-32-5p, 92-b-3p, and 301a-3p positively regulating cellular proliferation and downregulation of miR-5571, -4670 negatively regulating smooth muscle proliferation in EVs from HIV-PH patients. This increase in miR-301a-3p and decrease in miR-4670 were negatively correlated with the CD4 count and FEV1/FVC ratio, and positively correlated with viral load. Collectively, this data suggest the association of alterations in the miRNA cargo of circulating EVs with HIV-PH.

Association of Altered Ratio of Maternal Folic Acid and Vitamin B12 during Pregnancy with Newborn Birth Weight, Head Circumference, and Chest Circumference.

OBJECTIVE: This study evaluated the effect of an altered ratio of maternal RBC folate (MRF) to serum vitamin B12 (MB12) on pregnancy and newborn outcomes. METHODS: Blood samples were collected from pregnant women and the umbilical cord at the time of delivery. Estimations of RBC folate and serum vitamin B12 from maternal and cord blood samples and total homocysteine (HCY) were performed. Maternal and newborn anthropometric parameters like placental weight (PW), head circumference (HC), chest circumference (CC), and body weight (BW) were measured in offsprings after birth. We stratified the pregnant women into six groups (a) vitamin B12 normal and folic acid normal (BNFN)-control group, (b) vitamin B12 normal and folic acid elevated (BNFE), (c) vitamin B12 normal and folic acid deficient (BNFD), (d) vitamin B12 deficient and folic acid normal (BDFN), (e) vitamin B12 deficient and folic acid elevated (BDFE) and (f) vitamin B12 deficient and folic acid deficient (BDFD) based on their levels of RBC folate (MRF) and vitamin B12 (MB12). The expression of the one-carbon metabolism genes (methionine synthase (MS), glycine N-methyltransferase (GNMT), and cystathionine ß-synthase (CBS) was also studied in placental tissue by using real-time PCR. RESULTS: Cord blood RBC folate was significantly reduced in groups BDFE and BDFD as compared to the control group (BNFN). The cord blood vitamin B12 levels were also reduced in the BDFE group as compared to the BDFD. All the newborn parameters viz. PW, HC, CC, and BW, were reduced in the altered MRF/MB12 ratio (low & high vs. normal ratio). Total HCY was significantly elevated in the groups with (BDFE & BDFN) an imbalance of maternal RBC folate and serum vitamin B12 as compared to the control group. Downregulation of one-carbon metabolism genes like MS (p < 0.001), GNMT (p < 0.05), and CBS (p < 0.01) in placental tissue was observed in the high MRF/MB12 ratio group as compared to the normal ratio group. A strong positive correlation was also observed between MRF, MB12, and newborn parameters. CONCLUSIONS: The altered ratio of folate to vitamin B12 in the maternal blood is associated with adverse growth and development of the newborn.

Integrative analysis of the lncRNA-miRNA-mRNA interactions in smooth muscle cell phenotypic transitions.

Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown. Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs. Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236. Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.

Pre-natal dietary imbalance of folic acid and vitamin B12 deficiency adversely impacts placental development and fetal growth.

INTRODUCTION: The common practice of supplementing folic acid during pregnancy and the absence of such guidelines for vitamin B12 lead to an imbalance of these vitamins, especially in developing countries like India, where many women are vitamin B12 deficient. METHODS: The present study was designed to explore the effect of low vitamin B12 in combination with different levels of folic acid in the parental diet on fetal growth parameters and maternal reproductive performance in a transgenerational manner. The reversibility of these effects was studied by shifting the mice to a regular diet in the F1 generation in the case of transient groups and continued on the same diet in the sustained groups after the dietary exposure in the F0 generation. RESULTS: Vitamin B12 deficiency and different levels of folic acid resulted in the decreased placental and fetal weight of the F1 generation. Surprisingly, a decreased placental weight, low fetal weight, and reduced crown-rump length and head circumference were observed in F2 fetuses of vitamin B12 deficient with folate over-supplemented (BDFO) transient group, i.e. when F1 mice were shifted to normal diet conditions. Reduced follicles in ovaries and alteration in placental pathology in all the F0 groups and BDFO of the F1 transient group were also seen. DISCUSSION: Overall, the study revealed that dietary imbalance of vitamin B12 and folic acid, particularly B12 deficiency with over-supplemented folic acid, negatively affects placental and fetal development and maternal reproductive performance. Such effects are passed on to the next generation too.

High dietary folate and low vitamin B12 in parental diet disturbs the epigenetics of imprinted genes MEST and PHLDA2 in mice placenta.

To elucidate the dietary effects of vitamin B12 and folic acid on fetal and placental epigenetics, different dietary combinations of folic acid and low vitamin B12 (four groups) were fed to the animals (C57BL/6 mice), and mating was carried out within each group in the F0 generation. After weaning for 3 weeks in the F1 generation each group is divided into two sub-groups, while one group of mice was continued on the same diet (sustained group), the other was shifted to a normal diet (transient group) for 6-8 weeks (F1). Mating was carried out again within each group, and on day 20 of gestation, the maternal placenta (F1) and fetal tissues (F2) were isolated. Expression of imprinted genes and various epigenetic mechanisms, including global and gene-specific DNA methylation and post-translational histone modifications, were studied. Evaluation of mRNA levels of MEST and PHLDA2 in placental tissue revealed that their expression is maximally influenced by vitamin B12 deficiency and high folate conditions. The gene expression of MEST and PHLDA2 was found significantly decreased in the F0 generation, while over-expression was seen in BDFO dietary groups of F1 generation. These dietary combinations also resulted in DNA methylation changes in both generations, which may not play a role in gene expression regulation. However, altered histone modifications were found to be the major regulatory factor in controlling the expression of genes in the F1 generation. The imbalance of low vitamin B12 and high folate leads to increased levels of activating histone marks, contributing to increased gene expression.

Impact of human immunodeficiency virus on pulmonary vascular disease.

With the advent of anti-retroviral therapy, non-AIDS-related comorbidities have increased in people living with HIV. Among these comorbidities, pulmonary hypertension (PH) is one of the most common causes of morbidity and mortality. Although chronic HIV-1 infection is independently associated with the development of pulmonary arterial hypertension, PH in people living with HIV may also be the outcome of various co-morbidities commonly observed in these individuals including chronic obstructive pulmonary disease, left heart disease and co-infections. In addition, the association of these co-morbidities and other risk factors, such as illicit drug use, can exacerbate the development of pulmonary vascular disease. This review will focus on these complex interactions contributing to PH development and exacerbation in HIV patients. We also examine the interactions of HIV proteins, including Nef, Tat, and gp120 in the pulmonary vasculature and how these proteins alter the endothelial and smooth muscle function by transforming them into susceptible PH phenotype. The review also discusses the available infectious and non-infectious animal models to study HIV-associated PAH, highlighting the advantages and disadvantages of each model, along with their ability to mimic the clinical manifestations of HIV-PAH.

Circulating Cell-Free Nucleic Acids as Epigenetic Biomarkers in Precision Medicine.

The circulating cell-free nucleic acids (ccfNAs) are a mixture of single- or double-stranded nucleic acids, released into the blood plasma/serum by different tissues via apoptosis, necrosis, and secretions. Under healthy conditions, ccfNAs originate from the hematopoietic system, whereas under various clinical scenarios, the concomitant tissues release ccfNAs into the bloodstream. These ccfNAs include DNA, RNA, microRNA (miRNA), long non-coding RNA (lncRNA), fetal DNA/RNA, and mitochondrial DNA/RNA, and act as potential biomarkers in various clinical conditions. These are associated with different epigenetic modifications, which show disease-related variations and so finding their role as epigenetic biomarkers in clinical settings. This field has recently emerged as the latest advance in precision medicine because of its clinical relevance in diagnostic, prognostic, and predictive values. DNA methylation detected in ccfDNA has been widely used in personalized clinical diagnosis; furthermore, there is also the emerging role of ccfRNAs like miRNA and lncRNA as epigenetic biomarkers. This review focuses on the novel approaches for exploring ccfNAs as epigenetic biomarkers in personalized clinical diagnosis and prognosis, their potential as therapeutic targets and disease progression monitors, and reveals the tremendous potential that epigenetic biomarkers present to improve precision medicine. We explore the latest techniques for both quantitative and qualitative detection of epigenetic modifications in ccfNAs. The data on epigenetic modifications on ccfNAs are complex and often milieu-specific posing challenges for its understanding. Artificial intelligence and deep networks are the novel approaches for decoding complex data and providing insight into the decision-making in precision medicine.

Effects of altered maternal folate and vitamin B12 on neurobehavioral outcomes in F1 male mice.

Maternal folate and vitamin B12 status during pregnancy may influence development of central nervous system (CNS) in the offspring. Very little attention has been paid to understand the combined effects of both the vitamins during pregnancy. The present study was designed to evaluate the biochemical and behavioral outcomes following alterations in folate and vitamin B12 levels in C57BL/6 mice. The female mice were fed with different combinations of folate and vitamin B12 whereas; males were fed with normal diet for 4 weeks. The mice were mated and the pregnant mice received the same diets as before pregnancy. The F1 male mice were further continued on maternal diet for 6 weeks following neurobehavioral and biochemical assessment. The body weight of the F1 male mice was significantly decreased in the mice that received folate and vitamin B12 deficient diet. Altered cognitive functions were observed in the folate and B12 deficient F1 male mice as assessed by Morris water maze and novel object recognition tests. Spontaneous locomotor activity was decreased in F1 male mice fed with folate and B12 deficient diets. Elevated homocysteine levels and decreased hydrogen sulfide levels were also observed in the brain of F1 male mice on folate and B12 deficient diets. However, GSH and GSSG levels were increased in the brain of the animals supplemented with folate deficient diet with different combinations of B12. The study suggests that exposure of female mice to folate and vitamin B12 deficiency during pregnancy effects in-utero development of fetus, which further leads to behavioral anomalies in adult life and is sufficient to cause impaired cognitive behavior in the subsequent generation. Thus, elucidating the role and importance of maternal dietary folate and B12 ratio during pregnancy.

Effect of imbalance in folate and vitamin B12 in maternal/parental diet on global methylation and regulatory miRNAs.

DNA methylation, a central component of the epigenetic network is altered in response to nutritional influences. In one-carbon cycle, folate acts as a one-carbon carrier and vitamin B12 acts as co-factor for the enzyme methionine synthase. Both folate and vitamin B12 are the important regulators of DNA methylation which play an important role in development in early life. Previous studies carried out in this regard have shown the individual effects of these vitamins but recently the focus has been to study the combined effects of both the vitamins during pregnancy. Therefore, this study was planned to elucidate the effect of the altered dietary ratio of folate and B12 on the expression of transporters, related miRNAs and DNA methylation in C57BL/6 mice. Female mice were fed diets with 9 combinations of folate and B12 for 4 weeks. They were mated and off-springs born (F1) were continued on the same diet for 6 weeks post-weaning. Maternal and fetal (F2) tissues were collected at day 20 of gestation. Deficient state of folate led to an increase in the expression of folate transporters in both F1 and F2 generations, however, B12 deficiency (BDFN) also led to an increase in the expression in both the generations. B12 transporters/proteins were found to be increased with B12 deficiency in F1 and F2 generations except for TC-II in the kidney which was found to be decreased in the F1 generation. miR-483 was found to be increased with all conditions of folate and B12 in both F1 and F2 generations, however, deficient conditions of B12 led to an increase in the expression of miR-221 in both F1 and F2 generations. The level of miR-133 was found to be increased in BDFN group in F1 generation however; in F2 generation the change in expression was tissue and sex-specific. Global DNA methylation was decreased with deficiency of both folate and B12 in maternal tissues (F1) but increased with folate deficiency in placenta (F1) and under all conditions in fetal tissues (F2). DNA methyltransferases were overall found to be increased with deficiency of folate and B12 in both F1 and F2 generations. Results suggest that the dietary ratio of folate and B12 resulted in altered expression of transporters, miRNAs, and genomic DNA methylation in association with DNMTs.

Epigenetic modifications at DMRs of placental genes are subjected to variations in normal gestation, pathological conditions and folate supplementation.

Invasive placentation and cancer development shares many similar molecular and epigenetic pathways. Paternally expressed, growth promoting genes (SNRPN, PEG10 and MEST) which are known to play crucial role in tumorogenesis, are not well studied during placentation. This study reports for the first time of the impact of gestational-age, pathological conditions and folic acid supplementation on dynamic nature of DNA and histone methylation present at their differentially methylated regions (DMRs). Here, we reported the association between low DNA methylation/H3K27me3 and higher expression of SNRPN, PEG10 and MEST in highly proliferating normal early gestational placenta. Molar and preeclamptic placental villi, exhibited aberrant changes in methylation levels at DMRs of these genes, leading to higher and lower expression of these genes, respectively, in reference to their respective control groups. Moreover, folate supplementation could induce gene specific changes in mRNA expression in placental cell lines. Further, MEST and SNRPN DMRs were observed to show the potential to act as novel fetal DNA markers in maternal plasma. Thus, variation in methylation levels at these DMRs regulate normal placentation and placental disorders. Additionally, the methylation at these DMRs might also be susceptible to folic acid supplementation and has the potential to be utilized in clinical diagnosis.