Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy
Study of the molecular variation in pre-eclampsia placenta based on micro-Raman spectroscopy.
Five pregnant women with pre-eclampsia from Nanfang hospital were selected as study group whose average age is 28.5 years and 38 ± 2 weeks gestation. The same period of healthy pregnant women, whose average age is 27.6 years and pregnant 39 ± 1 weeks, as control group (n = 5). The normal and pre-eclamptic placental tissues are detected by micro-Raman spectroscopy with the spectrum resolution of 1 cm−1.
We find that the protein structure of α-helix, β-pleated sheet and β-turn is overlying in pre-eclamptic placenta, which lead to a disorder of protein structure. The Raman peaks assigned to tryptophan indole ring and phenylalanine in pre-eclamptic placental tissue are more higher than that in normal tissue.
Results suggest that the ordered structures of the main chain in protein molecules are reduced significantly, and the amino acid of side chains is damaged obviously. And a principal component analysis is used to classify the Raman spectra between normal and pre-eclamptic placental tissues. This study presents that Raman spectroscopy has a great potential on the mechanism research and diagnosis of placental lesions.
KeywordsMolecular variation Pre-eclampsia placenta Micro-Raman spectroscopy Principal component analysis Scores plots
Pre-eclampsia (also known as pregnancy-induced hypertension, PIH) is a disorder that generally develops late in pregnancy and is characterized by a sudden onset of high blood pressure, edema and protein in the urine . However, the immunity, impaired vascular endothelial cells and insulin resistance etiology in this disease are unclear . Researchers are interested in the etiology and pathology of pre-eclampsia. Turner et al.  observed that the concentration of tyrosine, histidine, and phenylalanine in pre-eclampsia placenta was higher than normal tissue. And Raouf et al.  reported that there was a decreased intensity at the protein bands from the Fourier spectra of lyophilized serum samples. There were some conflicting views in the previous studies [3, 4]. Therefore, an in-depth research on the molecular and protein variation of pre-eclampsia is urgently needed for scientists.
Confocal micro-Raman spectroscopy, which depends on polarizability change of an oscillating molecule, has proven extremely versatile and has led to a vast array of applications across the disciplines of chemistry, physics, biology, biomedicine, engineering, and archeology. With an insensitivity to the presence of water, Raman spectroscopy is a particularly attractive technique for life sciences. In addition to these, compared with other optics technologies including second harmonic generation, Fourier transform infrared spectroscopy (FTIR), and multiphoton microscopy, Raman spectroscopy requires simple sample preparation and contains abundant information [5, 6, 7, 8]. With the help of these superiorities, Raman spectroscopy has been employed to detect the damage of gestational diabetes on placenta . However, there were few reports about placenta in pre-eclampsia. In this study, the micro-Raman spectra were employed to characterize the molecular variation in pre-eclamptic placenta tissues.
Materials and methods
Sample collection and preparation
Informed consent for the study was obtained from all the women concerned, and approval was given by the Ethics Committee of the Faculty of Medicine, Nanfang Hospital, Southern Medical University. In this study, five pregnant women with pre-eclampsia from Nanfang hospital were selected as study group whose average age is 28.5 years and 38 ± 2 weeks gestation. The same period of healthy pregnant women, whose average age 27.6 years and pregnant 39 ± 1 weeks, as control group (n = 5). All of the placentas were obtained immediately after cesarean deliveries. There were no other obstetric complications in the two groups of pregnant women.
Tissue samples were placed on a silicon slice for measurement of Raman spectroscopy because there were no extra Raman peaks for the silicon slice in the fingerprint spectrum region from 700 to 1,800 cm−1. The Raman spectra were acquired using a Renishaw (New Mills, UK) inVia confocal micro-Raman spectroscopy system. The samples were excited by 785 nm laser and after attenuation through prisms and filter, the power of the laser exposed on the samples was 9 mW. Spectra were obtained from tissues with a 20× optimized objective and the signal was integrated for five times over a spectral range of 700–1,800 cm−1. Peak frequencies are calibrated with the silicon at 520 cm−1. For each sample, at least 20 Raman spectra are obtained. All the data are collected under the same conditions. Tissue samples were placed on a silicon slice for Raman measurement. In order to compare the related spectra changes, the phenylalanine band (1,004 cm−1) was chosen to normalize the spectra. The final Raman spectra are baseline corrected by the software R 2.8.1, and smoothed, normalized, and averaged by ORIGIN PRO 8.5 (OriginLab Corporation, Northampton, MA, USA), together with the Raman spectroscopic software WIRE 3.2.
Raman shift and the tentative assignment of normal placenta
Amide I protein
By comparing the Raman spectra of normal and pre-eclamptic placental tissues, it was clear that the characteristic spectral line of amide I at 1,663 cm−1 from normal placenta assigned to α-helix structure, but peaks at 1,662 and 1,640 cm−1 in pre-eclamptic placental tissue showed a superposition composed by α-helix, β-pleated sheet and β-turn which led to a disorder of protein structure . Meanwhile, the Raman spectra also showed variations of amino acid residue on the protein side chain. In pre-eclamptic placenta, we observed significant enhanced spectral lines of tryptophan indole ring and phenylalanine and some special lines at 1,399 and 1,640 cm−1 which cannot be found in normal placental tissue, which shows an addition of the content of amino acid residue in pre-eclamptic placenta. The vasoactive substances synthesized and secreted by hypoxic–ischemic placenta (such as soluble Fms-like tyrosine kinase 1, cytokines, angiotensin II type 1 receptor activating antibody and thromboxane) entered into the maternal blood circulation and caused vascular endothelial cell dysfunction, which can further promote the release of vasoactive substances to decrease the activity of NO and increase reactive oxygen species and free radicals. Oxidation changed the main chain conformation and injured the side chain groups of protein which presented characteristic enhanced spectral lines of phenylalanine at 1,005, 1,605 and 1,620 cm−1 and those amines with activity side chain such as tryptophan indole ring at 758 and 1,585 cm−1 [13, 14].
In this study, the results show that: (1) the protein structure of α-helix, β-pleated sheet and β-turn is overlying in pre-eclamptic placenta, which lead to a disorder of protein structure. (2) The Raman peaks assigned to tryptophan indole ring and phenylalanine in pre-eclamptic placental tissue are more higher than that in normal tissue. It suggests that the ordered structures of the main chain in protein molecules are reduced significantly, and the amino acid of side chains is damaged obviously. (3) The PCA could give us a useful help on distinguishing the Raman spectra between normal and pre-eclamptic placental tissues. And the Raman spectroscopy presents a great potential on the mechanism research and diagnosis of placental lesions.
This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20134407120003), the PhD Startup Fund of Natural Science Foundation of Guangdong Province, China (No. S2013040016223), the National Natural Science Foundation of China (No. 61275187, 11374107), and the Young Teachers Nurturing Fund of South China Normal University (2012KJ020) and the Horizontal Topic Research Project of SENSING.
Conflict of interest
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