The mass spectrometry-based precision assay for B-vitamins in erythrocytes is a new technology developed by Prof. Hongjun Shi, an independent principal investigator from Westlake’s School of Life Sciences. The team has recently received a pre-A round of funding reaching tens of millions of yuan, going from the laboratory to the market.

His lab is the first diagnostic institution engaged in mass spectrometry-based assays of metabolic small molecules in Westlake’s agenda for the commercialization of research outcomes. It is also the first organization in the world that has realized an accurate mass spectrometry-based assay on the metabolic capacity of B vitamins in erythrocytes.

With this new technology, doctors are able to know the exact levels of folic acid in a woman preparing for pregnancy, which allows more accurate dosing.

A Question for Clinicians: How Much Folic Acid Should Be Taken?

B vitamins are essential water-soluble nutrients required by the human body. Deficiency of B vitamins is associated with symptoms or diseases such as birth defects, recurrent miscarriages, pregnancy hypertension, and preeclampsia. Folic acid, as one of the B vitamins, has been proven by scientific studies as an effective pre-pregnancy supplement. It can reduce the risk of birth defects, such as neural tube defects and congenital heart disease. Therefore, taking folic acid has been included in the guidelines written by professional organizations in various countries as an important method to prevent birth defects.

In daily life, folic acid is widely found in foods made of plants and meats, especially in green leafy vegetables. However, natural folic acid is extremely unstable, and the human body does not really get much folic acid from food. Moreover, as the body cannot synthesize folic acid by itself, we have to rely on extra supplementation of folic acid. According to a large sample of data, the current recommended intake for pre-pregnant women is 0.4-0.8 mg per day.

But does this standard apply to everyone? Different genetic factors, dietary habits, and lifestyles can affect the level of folic acid in each individual body. Will consuming the same dose of folic acid cause some people to overdose and others to underdose?

But the rapid and accurate assay of folic acid is a headache for clinical practices.

Clinical assays of folic acid are mainly performed on blood serum or red cells. Although the serum-based assay is more mature, it mainly reflects recent intake of folic acid, which is easily influenced by diet and therefore quite unstable.

Compared with folic acid in blood serum, folic acid in red cells reflects long-term storage of folate in the body. It has therefore been recognized as a better indicator of folate. However, unlike free-state serum folate, intracellular folate requires multiple steps such as cell purification and lysis, polyglutamic hydrolysis, and antioxidant protection. It is therefore technically difficult to enable folate assay based on red cells.

The microbiological method was previously recognized by the clinical nutrition community as the gold standard for erythrocyte-based assay of folic acid. But the method is time-consuming and requires high aseptic operation, which is not quite applicable to clinical practices.

In addition, deficiencies of B vitamin coenzymes such as B2, B3, and B6 may also affect the efficiency of folic acid metabolism. To fully evaluate folate levels and their metabolic function, the levels of these B vitamins in the human body have to be tested at the same time.

The Finding of Hongjun Shi: Identifying Folic Acid with Mass-to-Charge Ratio

Prof. Hongjun Shi

Unexpectedly, Shi from the Westlake Cardiac Development and Regeneration Laboratory undertook this project on folate assay.

With a research interest in the pathogenesis of congenital heart disease, Shi used to work at the Victor Chang Cardiac Research Institute as a postdoctoral research fellow at the Department of Developmental and Stem Cell Biology Research. He also worked as a part-time lecturer at St. Vincent's School of Medicine, the University of New South Wales.

Because of his research into congenital heart disease, Shi communicated with many doctors in maternity hospitals and found that many of them were confused about the dosage of folic acid. Paradoxically, B vitamins such as folic acid and niacin are inextricably linked to the development of congenital heart disease.

"Theoretically, we should be able to find a better way," said Shi. So he decided to start designing experiments that would solve this problem for doctors.

"Each small molecule has a specific mass-to-charge ratio. If we can identify this ratio in a substance, we can know what kind of substance it is,” said Shi. His research team innovatively applied a mass spectrometry-based assay to detect folic acid in isolated red cells. "B vitamins have different forms in the body, and the mass spectrometry equipment can clearly distinguish the mass-to-charge ratios of different B vitamins, thus accurately measuring their amount in the body."

Different B vitamin coenzymes vary in their small molecule structures and physicochemical properties, so the question of how to achieve compatibility in extraction was one of the biggest challenges. Finally, they developed a new stable extraction method for B vitamins in erythrocytes to achieve simultaneous extraction and detection of B2, B3 (niacin), B6, and folic acid within 30 minutes.

"We have now completed comprehensive folate function testing in tens of thousands of samples from across the country. The results are traceable to the WHO International Standard for Folate in Whole Blood Haemolysate (95/528), which further confirmed the accuracy of the method," said Shi.

Through sample analysis, the team found that the average folate level (around 650 nmol/L) of pregnant Chinese women was equal to that in the United States before mandatory folic acid supplementation, but far from the level after folic acid supplementation (1,150 nmol/L). Shi said, "The national nutrition plan announced in 2017 stated that the folic acid deficiency rate among women at the age for childbearing should be reduced to less than 5%, but according to our test results, 10% to 30% of pregnant Chinese women don’t have sufficient folic acid in their bodies, and the rate of folic acid deficiency shows a regionally increasing trend from the south, to the north, and then to the western region."

The distribution of folate levels in Chinese women of childbearing age, based on mass spectrometry-based assay of folic acid in erythrocytes.

For women who are preparing for pregnancy, they can now simply go to the hospital to take a blood test and receive the most reliable report of their folic acid levels, which allows their doctors to provide personalized medication guidance.

A Scientist’s Further Exploration: Genetic Testing for Congenital Heart Disease

Based on this new technology, Vito Diagnostics (Hangzhou) has already started their business operation with its headquarters in Xihu District. For his further research, Shi has included the assay of niacin, one of the B vitamins, in his to-do list. In previous studies, Shi and his team found that niacin deficiency could also lead to multi-organ birth defects including congenital heart disease.

"Our experiments are currently performed on mice, and we need to collaborate with more hospitals to further validate our previous hypothesis," said Shi.

Shi's ultimate goal is to develop a series of innovative molecular diagnostic technologies focusing on complex diseases such as birth defects, tumors, cardiovascular and cerebrovascular diseases, and genetic and metabolic diseases for clinical services such as individualized diagnosis and precise medication guidance, including genetic testing for congenital heart disease.

Mouse hearts in Shi’s Lab

The incidence of congenital heart disease is as high as 1%, meaning that one out of every 100 newborns has congenital heart disease. The causes of the disease are so complex that we only know 20% of them, and the remaining 80% are still an unsolved mystery.

From 2018 to date, Shi's team has identified several groups of genes that might lead to

congenital heart disease with their simultaneous mutations, providing unprecedented evidence for a polygenic etiology in the disease. "If these findings can be applied clinically, they can provide an effective means for the prevention and early molecular diagnosis of birth defects,” said Shi.

Shi is very grateful to those clinicians who raised the question in the first place. “We do not carry out research just for the research itself. Our ultimate goal is to benefit mankind,” said Shi.