Effect of 405 nm low intensity irradiation on the absorption spectrum of in-vitro hyperlipidemia blood

1.Introduction

Dyslipidemia is one of common diseases in the general population. Lipid and lipoprotein abnormalities are regarded as a modifiable risk factor for cardiovascular disease due to their influence on atherosclerosis [1, 2, 3]. It is crucial to improve lipid levels in the body for the treatment of hyperlipidemia. The therapies were reported be effective in clinical practice, such as laser therapy [4, 5, 6], hypolipidemic drugs [7]. Low-level laser therapy (LLLT) has been found to modulate various biological processes without side effects. Low level laser of 650 nm showed an effect on total number of erythrocytes, HCT and red blood cells aggregation [8]. Low intensity laser improve microcirculation, rheological properties and blood lipid that might be related with erythrocytes aggregation and deformability [9]. In earlier reports, low-intensity laser radiation of blood changed red cell count in elderly patients with coronary heart disease and activated the blood antioxidant system [10, 11]. Low-intensity laser radiation in essential hypertension has the efficiency to increase oxygen content and stimulate microcirculation [12, 13].

In addition, low-intensity laser radiation can break off some molecular bonds in glucose of blood, thus glucose alcohol decomposition produces ATP energy, and red cell obtain enough energy, as a result, its deformability increases. Meanwhile, there is a decrease in platelet aggregation which originally suffered an increase, due to increased levels of serum oxidized LDL [14]. Chi et al. [15] proved firstly the effect of intranasal red laser in treating hyperlipidemia by ILILT (Intranasal Low Intensity Low Laser Therapy) with LGAL diode at 650 nm and 5 mw. Nesreen found that a significant improvement in lipid profile after adding blue laser (405 nm) blood irradiation therapy [16]. However the lack of certainty on the mechanism of improving blood lipid profiles following LLLT will tend to slow down the widespread adoption of these techniques [17].

In the present study, the effect on absorption spectrum for whole blood and red blood cells after 405 nm low-level laser irradiation (LLLI) was analyzed to demonstrate efficacy of low level laser therapy for blood lipid level of hyperlipidemia in vitro. Effective factors such as red blood cell count (RBC), Red blood cell specific volume (HCT), hemoglobin and gender were considered. This study intends to provide new ideas and methods to improve the blood lipid level of hyperlipidemia.

2.Materials and methods

3.Results

3.1The absorption spectra of the whole blood

The absorption spectrum both control group and experimental group was measured by ultraviolet-visible spectrophotometry to study cholesterol influence on the characteristic absorption peak in blood. Intensive absorption peaks concentrated on 200–300 nm. In visible wavelengths of 400 nm–760 nm, there were several characteristic peaks at 416 nm, 544 nm, 578 nm according to the result of two groups. The absorption spectra of normal human serum and hypercholesterolemia serum were obtained in Lan et al.’s study [18, 19]. The result indicated the absorptivity of hypercholesterolemia serum was higher than that of normal serum. Hypercholesterolemia serum had three absorption peaks locating on 578 nm, 542 nm and the strongest one 416 nm. Compared to the control group, there was no difference of characteristic peaks in hyperlipidemia group. Therefore, high-CHO level in blood had no significant effects on characteristic absorption peaks. The absorption spectra of two samples from control group and high-CHO group were presented in Fig. 1.

Figure 1.

The absorption spectra of two whole blood samples from control group (CHO: 3.33 mmol/L) and high-CHO group (CHO: 7.31 mmol/L).

Characteristic absorption peaks of the whole blood had little difference between control group and high-CHO group. Whereas the absorbance of characteristic peaks showed statistic difference (P< 0.05) in visible wavelengths of 400 nm–760 nm, in which the absorbance of 416 nm in High-CHO group was significantly higher than control group. The result is graphically displayed in Fig. 2.

3.3Lipid profiles and absorbance

The molar concentration of lipid profiles (CHO and TG) in two blood samples was measured and the concentration of TG was 1.07 ± 0.27 mmol/L in control group and 2.13± 0.88 mmol/L in high-CHO group. The absorbance of characteristic absorption peaks (416 nm, 544 nm, 578 nm) summarized in Table 2.

Table 2

The absorbance of lipid profiles (CHO and TG) on absorption peaks

Group	Sample	CHO	TG	416 nm	544 nm	578 nm
		(mmol/L)	(mmol/L)
Control	Whole blood	4.636 ± 0.84	1.07 ± 0.27	2.43 ± 0.95	0.96 ± 0.33	0.98 ± 0.34
	Erythrocyte			2.97 ± 0.56	1.1 ± 0.20	1.12 ± 0.19
High-CHO	Whole blood	8.032 ± 1.04	2.13 ± 0.88	3.10 ± 0.96	1.11 ± 0.24	1.14 ± 0.25
	Erythrocyte			3.41 ± 0.93	1.22 ± 0.24	1.25 ± 0.25

Table 3

Comparison of RBC, HCT and hemoglobin

Group	CHO (mmol/L)	Hemoglobin (g/L)	RBC (1012/L)	HCT
Control	4.636 ± 0.84	139.4 ± 18.81	6.129 ± 1.34	0.423 ± 0.05
High-CHO	8.032 ± 1.04	133.8 ± 6.58	8.153 ± 1.33	0.408 ± 0.02

Figure 2.

Main absorption peaks (416 nm, 544 nm, 578 nm) from whole blood sample in hyperlipidemia and control groups.

Figure 3.

The absorption spectra of two erythrocyte samples from control group (CHO: 3.33 mmol/L) and high-CHO group (CHO: 7.31 mmol/L).

Figure 4.

Main absorption peaks (416 nm, 544 nm, 578 nm) from erythrocyte sample in hyperlipidemia and control groups.

3.5RBC, HCT, hemoglobin and gender

RBC, HCT and hemoglobin were considered as the factors of influencing visible absorption. To eliminate the interferences, RBC, HCT and hemoglobin from High-CHO group was in contrast to control group. There was no significant difference (> 0.05) for HCT and hemoglobin. However, there existed significant statistical difference (< 0.05) for RBC, the density of erythrocyte had positive influence on the absorbance in high-CHO group. For this reason, a conclusion can be drawn that the absorbance difference of absorption peaks was not caused by hemoglobin and HCT, except for RBC. All results were given in Table 3.

To study the gender distinctions, the sample in high-CHO group was regrouped. In male group, the mean CHO is 8.21 ± 1.42 mmol/L, slightly higher than female (7.86 ± 0.58 mmol/L). The absorbance of characteristic absorption peaks was compared respectively in the erythrocyte and whole blood. The result was shown in Table 4 and sexual differences didn’t exist in absorbance of characteristic absorption peaks in the blood samples.

Table 4

The absorbance of gender distinction

Gender	CHO	Whole blood			Erythrocyte
–	–	416 nm	544 nm	578 nm	416 nm	544 nm	578 nm
Male	8.21 ± 1.42	3.03 ± 0.78	1.13 ± 0.2	1.16 ± 0.21	3.191 ± 1.16	1.15 ± 0.19	1.18 ± 0.19
Female	7.86 ± 0.58	3.1 ± 1.2	1.08 ± 0.3	1.11 ± 0.30	3.63 ± 0.71	1.29 ± 0.29	0.3 ± 0.3

Figure 5.

The absorbance of main absorption peaks (416 nm, 544 nm, 578 nm) before and after LLLT (a) the whole blood sample (b) the erythrocyte sample.

4.Discussion

In this experiment, the influence on hyperlipidemia in vitro after low-intensity laser irradiation was reflected by characteristic absorption spectrum. The absorption spectrum of whole blood was obviously lower than which of hyperlipidemia. The overall absorbance of 300–700 nm in high-CHO group was higher due to the existence of high CHO and TG. And the absorption peak of the most absorbance was at 416 nm, but the absorption peaks at 544 nm and 578 nm was not conspicuous. In Zhao et al.’s study, there is a clear absorption peak at 414 nm in the absorption spectrum of the blood serum with hypercholesterolemia [20].

To explore the hypercholesterolemia influence on erythrocyte, the absorption spectrum of both the erythrocyte and whole blood was determined. These results indicated that the characteristic absorption peaks (416 nm, 544 nm, 578 nm) of erythrocyte in the high-CHO group were higher than erythrocyte in the normal blood. After LLLT, the absorbance of the whole blood samples rose, whereas erythrocytes decreased. The concentration of extracellular cholesterol is elevated. It could be speculated that the cholesterol was separated from the erythrocyte. Cholesterol enrichment in erythrocytes results in decreasing deformability and fluidity, and changing the shape and rheological parameters of these cells [21]. It maybe leads to impairment of functional properties including membrane bound enzymes and rheological behaviors such as osmotic fragility that can promote atherosclerotic lesions [22, 23]. Cholesterol is mainly distributed on the outer layer of the red cell membrane with the ability of affecting membrane fluidity. The statins were used in the treatment of hyperlipidemia commonly. In atorvastatin therapy, the level of cholesterol in erythrocyte membranes was decreased and a significant change was observed in osmotic fragility values of the mixed-typed dyslipidemia group [24]. Similarly to atorvastatin therapy, LLLT had certain effect on erythrocyte membrane. The cholesterol absorption of erythrocyte is influenced on the lipid profiles of intracellular cholesterol or on the membrane.

The result of this study in Fig. 5 showed the absorbance of characteristic peaks in whole blood was elevated, meanwhile its absorbance of erythrocyte isolated from the wholes blood after low level laser irradiation was reduced. As a result, the erythrocyte releases the lipid profiles in the presence of laser irradiation. It can be speculated that low level laser irradiation affects the enzymes activity of lipid metabolism, improves the cholesterol balance of plasma and cytoplasm in erythrocyte, and decreases aggregation of the erythrocyte. In vitro studies suggested that LLLT increases fat loss from adipocytes by release of triglycerides [25]. However, much work still needs to be done at molecular level to study the mechanism.

In this study, there are some factors that need to be taken into concern. The presence of trisodium citrate is potentially a confounding factor. The absorption spectrum of anticoagulant is not clear; besides, citrate is a free radical scavenger and 405-nm laser irradiation generates free radicals which are eliminated by citrate precisely. So, it is difficult to explain whether trisodium citrate affects absorption spectrometry and the laser radiation on the assay. Some researches obtained the absorption spectrum of the whole blood, but the influence of anticoagulant has not been mentioned. Besides, the serum isolated from the whole blood after the laser irradiated was not detected by absorption spectrometry.