Abstract

This study conducted at Baquba Teaching Hospital from January to April 2024, investigated the lipid profiles of 75 participants (50 patients with Toxoplasma and 25 controls) aged between 50 and 60 years. Existing research suggests a potential link between Toxoplasma infection and alterations in lipid metabolism; however, specific effects on different lipid fractions are not well-understood. This study aimed to explore the association between Toxoplasma infection and changes in lipid profiles, particularly focusing on whether infection status correlates with variations in high-density lipoprotein (HDL) levels. Venous blood samples were collected pre-breakfast and processed to obtain serum, which was analyzed for lipid profiles. The findings indicated that lipid levels generally increased in Toxoplasma patients compared to controls, except for a decrease in HDL levels. These results suggest that Toxoplasma infection could influence lipid metabolism, potentially impacting cardiovascular risk. Further research is needed to understand the mechanisms underlying these changes and their clinical implications.

Highlights:

• Toxoplasma infection is linked to significant changes in lipid profiles.
• The study identifies a unique decrease in HDL levels among infected individuals.
• Implications suggest potential cardiovascular risks associated with Toxoplasma.

Keywords: Toxoplasma, Lipid Metabolism, Cardiovascular Risk, HDL Decrease

Introduction

The protozoan parasite infection One of the most prevalent parasite infections in humans and other warm-blooded animals is Toxoplasma gondii. It can be found all throughout the world, from Australia to Alaska. Approximately one-third of the human race has encountered this parasite [1]. Most adults are not very ill from it, but in congenitally infected children, blindness and mental retardation can occur; in individuals infected after birth, blindness can occur; and in immunocompromised individuals, the disease can be fatal. The two main risk factors for T. gondii infection are eating or drinking anything contaminated with oocysts and consuming raw or undercooked animal products.

1. Toxoplasma Gondii and Lipid Profile

Toxoplasma gondii, a member of the Apicomplexa eukaryotes, is the source of toxoplasmosis, an infection. Toxoplasmosis is a common disease that affects warm-blooded animalsand humans alike. The economy and public health are both seriously threatened by this protozoan parasites[2]. Because T. gondii infections can result ina variety ofclinical outcomes, include abortions, mental impairmente, hydrocephalus, retinochoroiditis, andeven death and With potentially fatal encephalitis in AIDS patients receiving organ transplants and immunosuppressive medication, they nevertheless represent a major risk topublic health [3], [4]. According to estimates, T,gondii infections impact between 30% and 65% of the world's population [5]. Human seroprevalence of T,gondiiin infections rises withage, but itis lower in colder climates and does not differ much between genders [6]. In Iraq, attention to Toxplasmosishas disease recently sharply increased as a result of multiple investigations [7], [8]. Many researchers have been interested in the role that serum biochemical parameters play in human infections with certain parasites. These measures comprise total protein, total globulin, total albumin, (LDL)high-density lipoprotein , (VLDL)very-low density lipoprotein , (LDL)low-density lipoprotein, triglycerides, and cholesterol levels [9]. Studies conducted in vitro showed that these parasites may proliferate in lipid, rich, serum-free medium [10]. Assessing the levels of specific biochemical markers in individuals who test positive or negative for Toxoplasma is the goal of this study.

Methods

A. Standard Kits

B. Equipment and Tools

C. Study Design

The study is designed on 75 person (50patients and 25 control ) with Toxoplasma at range (50-60 y ) between January 2024 and April 2024 in the (central care unit) of the Baquba Teaching Hospital (B.T.H), the total number inthis study include conservatives 40 men and35 women .

The patients groups include :

1. Gender (30 male and 20 female)

2. Age of patients groups (50-60 years)

D. Blood Sample Collection

Blood is taken from each participant early in the morning, before to breakfast. Venous blood samples ranging from 10 to 15 milliliters were obtained from both patients and controls, and the samples were stored in a gel tube at room temperature for fifteen minutes (anticoagulant not included). After that, the serum was split into four aliquots by centrifugation at 3000 xg for fifteen minutes, and it was refrigerated at -20 °C until it was needed for a biochemical analysis.

1. Determinations of Human Choleisterols

a. Principle

The following is the reaction strategy for the enzymatic technique as described by Allain et al. :

Cholesterols esters → Cholesterols+free fattyacids

cholesterols + O2 → Cholesten 4 one3+H2O2

2H2O2 + Phenols + PAP → Quienoneimine (pink) + 4 H 20

b. Reagent Preparations

Remove the aluminum cap with a non-sharp instrument. Quickly fill vial R1 with the contents of vial R2. Gently stir until fully dissolved. Vial R3: Ready used .

c . Assay Procedoure

1) Manaus Methods

Keep the reagent and the samples on room temperatures.

a) User validation is required for performances involving manual procedures.

b) Applications for Kenza and other applications are available upon request.

2. Determinations of Human LDL-Cholesterols

a. Principe

Low-density lipoproteins (LDL) are specifically precipitated by polyvinyl sulfate in whole serum, which is the basis for the separation procedure used in this technique, Centrifugation is used to settle the precipitant, and the clear supernata is then tested for residual cholesterol from the remaining lipoproteins (VLDL+ HDL). Calculating LDL-cholesterol involves deducting the sample's total cholesterol from the supernatant cholesterol fractions.

b. Reagents Preparations

Until the date of expiration indicated on the label, all of the kit chemicals remain stable. During use, keep the vials tightly closed, shielded from light, and free from contaminations.

c. Procedures

1) Precipitating

a. Samples and reagents should be brought on room temperatures.

b. Pipette in centrifuge tubes that have been labeled

c. Vortex, then let rest at room temperature for ten minutes.

d. You can centrifuge for two minutes at 12,000 rpm or for ten minutes at 6000 rpm.

e. For the purpose of measuring cholesterol, removed of aliquot from the supernatant.

2) Calorimetry

a. Allow the kit's and the cholesterol MR's components to come to room temperatures.

b. Set up two sets of tests so that the total cholesterols in the sample and the cholesterols that is still in the supernatant may be measured simultaneously. Observe the insert's directions for total cholesterols.

c. Pipette into tubes with labels

Under light protection from light, the color remains constant for at least half an hour

3. Determinations of Human Trigyclerides MR

a. Principle

The foundation of method1,2 is the enzymatic breakdown of serum or plasma triglyceride by lipoprotein lipase (LPL), which yields glycerol and free fatty acids (FFA). Adenousine triphosphate (ATP) phosphorylates glyceruol in the presence of glycerolkinase (GK), resulting in the formation of glycerol-3-phosphat (G-3-P) and adenousine diphousphate (ADP). Glycerophosphate Oxidase (GPO) oxidizes G-3-P to produce hydrogen peroxide and dihydroxyacetone phosphate (DHAP). When4-aminoanteipyrine (4-AA)and phenoul are coupled with hydrogen peroxide (H2O2) under the action of peroxidase (POD), a red chromogen is created that is proportionate to the amount of triglycerides present in the sample.

Trieglycerides + 3H2O → Glyceroul + 3FFA

Glyceroul+ATP → Glyceroul-3-P+ADP

Glyceroul-3-P+O2 → DHAP+H2O

4-AA+4Phenoul → Quinouneimeine+H2O

b. Reagents Preparations

Both the Standards and the Monoreagents are prepared for usage.

c. Procedurs

1) Let the samples and reagents come on room temperature.

2) Pipette into tubes that have been labeled

3) Stir, then leave the tubes for either five minutes at 37ºciuisi or fifteen minutes on room temperature (16–25ºC).

4) After calibrating the samples and standards on 500 nm, compares their absorbance (A) with the reagenet blank.

Result and Discussion

A. Lipid Profiles and Glucose Biomarkers Among Study Groups

Patients with toxoplasmosis exhibited significantly higher mean levels across multiple parameters compared tohealthy controuls. Specifically, inpatient with toxoplasmosis, themean levels for glucose were 179.74 ± 14.33 mg/dL, compared to 111.68 ± 8.68 mg/dL in healthy controls (P = 0.001). Triglycerides (TG) levels were 204.88 ± 33.92 mg/dL in patients and 122.88 ± 29.14mg/dLin controuls (P~0.001). Tutal cholesterols (Total Ch) levels showed a mean of 267.90 ± 20.66 mg /dLin patient and 169.08 ± 17.63mg /dLin controul (P = 0.001). HighDensity Lipouprotein (HDL) level was significantly lowerin patient (13.92 ± 3.87 mg/dL) compared to controls (35.96 ± 3.01 mg/dL) (P~0.001). (LDL)LowDensity Lipoiprotein levels were higher in patients (163.60 ± 17.98 mg/dL) compared to controls (118.84 ± 5.49 mg/dL) (P = 0.001). Very Low-Density Lipoprotein (VLDL) levels was higherin patients (40.98 ± 6.78 mg/dL) compared to controls (17.12 ± 3.81 mg/dL) (P = 0.001). These findings highlight the significant metabolic and lipid profile alterations associated with toxoplasmosis, as detailed in Figure 1.

Figure 1. Comparative Analysis of Laboratory Test Results in Patients with Toxoplasmosis and healthy Controls

Figure 2. Lipid Profiles and Glucose values for control and patient group

B. Blood Glucose

For glucose levels, patients with toxoplasmosis (N=50) had a mean of 179.74 mg/dL, compared to 111.68 mg /dLin healthy contruols (N=25) (P ~ 0.001). The findings were in agreement with Anastasia Poznyaket al. (2020),[11]

Figure 3. Glucose values for control and patient group

C. Triglycerides (T.G.)

Triglycerides (T.G.) levels were significantly higher in patients (mean = 204.88 mg/dL) compared to controls (mean = 122.88 mg/dL) (P = 0.001). This outcome agreed with that of Lambertini C.et al. [12] were seen to be rising in T.G.'s serum.

Figure 4. Triglycerides values for control and patient group

D. Total C holesterol s (T C)

Total cholesterols (TC) levels were also higher in patients (mean = 267.90 mg/dL) compared to controls (mean = 169.08 mg/dL) (P = 0.001) This outcome was in agreement with Maha Radhi Abeass, et al. were seen rising in the total cholesterols serum sample.

Figure 5. Total cholesteruol values for control and patient group

E. High Denesity Lipouprotein (HDL)

Patients had significantly lower levels of High-Denesity Lipouprotein (HDL) (mean~13.92 mg/dL) than controls (mean~35,96 mg/dL) (P~0.001). This outcome was in agreement with Maha Radhi Abeass,,et al. [12] were found to be declining in the HDL cholesterols serum level.

Figure 6. HDL cholesterols values for control and patient group

F. Low Denesity Lipouprotein (LDL)

Patients had greater mean levels of low-denesity lipouprotein (LDL) (163.60 mg/dL) than controls (mean = 118.84 mg/dL) (P = 0.001). This outcome was consistent with Lambertini C.,et al. [13] were shown to be rising in the LDL serum.

Figure 7. LDL cholesterols values for control and patient group

G. Very Low Denesity Lipouproteib (V.LDL)

Very,Low-Denesity Lipouprotein (V.LDL) level was higherin patients (meun~40.976mg/ dL) comepared to control (meun~17.12mg/ dL) (P~0.001). This outcome was consistent with Lambertini [14] were shown to be rising in the VLDL serum.

Figure 8. VLDL cholesterol values for control and patient group

Conclusion

1. The results observed increasing in (Glucose ) values with Toxoplasma Patients.

2. Increasing of Lipid profile ( Total cholesterol , LDL cholesterol , T.G and VLDL ) with Toxoplasma Patients.

3. The HDL cholesterol decreases with cases Toxoplasma Patients.

References

1. J. P. Dubey and C. P. Beattie, "Toxoplasmosis of Animals and Man," CRC, Boca Raton, FL, 1988.
2. S. A. Hosseini, A. Amouei, M. Sharif, S. Sarvi, L. Galal, J. Javidnia, A. S. Pagheh, S. Gholami, A. Mizani, and A. Daryani, "Human Toxoplasmosis: A Systematic Review for Genetic Diversity of Toxoplasma Gondii in Clinical Samples," Epidemiology & Infection, vol. 147, 2019.
3. T. N. Chegeni, S. Sarvi, M. Moosazadeh, M. Sharif, S. A. Aghayan, A. Amouei, Z. Hosseininejad, and A. Daryani, "Is Toxoplasma Gondii a Potential Risk Factor for Alzheimer's Disease? A Systematic Review and Meta-Analysis," Microbial Pathogenesis, vol. 137, 103751, 2019.
4. D. Anvari, M. Sharif, S. Sarvi, S. A. Aghayan, S. Gholami, A. S. Pagheh, S. A. Hosseini, R. Saberi, T. N. Chegeni, Z. Hosseininejad, and A. Daryani, "Seroprevalence of Toxoplasma Gondii Infection in Cancer Patients: A Systematic Review and Meta-Analysis," Microbial Pathogenesis, vol. 129, pp. 30-42, 2019.
5. Jg, M., and O. Liesenfeld, "Toxoplasmosis," Lancet, vol. 363, no. 9425, pp. 1965-76, 2004.
6. M. Siponen, P. M. Kinnunen, J. Koort, H. Kallio-Kokko, O. Vapalahti, A. M. Virtala, and P. Jokelainen, "Toxoplasma Gondii Seroprevalence in Veterinarians in Finland: Older Age, Living in the Countryside, Tasting Beef During Cooking and Not Doing Small Animal Practice Associated with Seropositivity," Zoonoses and Public Health, vol. 66, no. 2, pp. 207-215, 2019.
7. M. M. Assim and E. J. Saheb, "The Association of Severe Toxoplasmosis and Some Cytokine Levels in Breast Cancer Patients," Iraqi Journal of Science, pp. 1189-1194, 2018.
8. R. H. Nafal, H. S. Al-Warid, and H. J. Al-Sultan, "Seroprevalence of Toxoplasmosis in Patients with Chronic Liver Disease in Baghdad," Iraqi Journal of Science, pp. 1667-1672, 2019.
9. L. R. Portugal, L. R. Fernandes, V. S. P. Pedroso, H. C. Santiago, R. T. Gazzinelli, and J. I. Alvarez-Leite, "Influence of Low-Density Lipoprotein (LDL) Receptor on Lipid Composition, Inflammation, and Parasitism During Toxoplasma Gondii Infection," Microbes and Infection, vol. 10, no. 3, pp. 276-284, 2008.
10. C. Bisanz, O. Bastien, D. Grando, J. Jouhet, E. Maréchal, and M. F. Cesbron-Delauw, "Toxoplasma Gondii Acyl-Lipid Metabolism: De Novo Synthesis from Apicoplast-Generated Fatty Acids Versus Scavenging of Host Cell Precursors," Biochemical Journal, vol. 394, no. 1, pp. 197-205, 2006.
11. D. Bansal, H. S. Bhatti, and R. Sehgal, "Role of Cholesterol in Parasitic Infections," Lipids in Health and Disease, vol. 4, no. 1, p. 10, 2005.
12. A. Poznyak, A. V. Grechko, P. Poggio, V. A. Myasoedova, V. Alfieri, and A. N. Orekhov, "The Diabetes Mellitus–Atherosclerosis Connection: The Role of Lipid and Glucose Metabolism and Chronic Inflammation," International Journal of Molecular Sciences, vol. 21, no. 5, 2020.
13. A. Lambertini, A. Zannoni, N. Romagnoli, et al., "Expression of Proteinase-Activated Receptor 2 During Colon Volvulus in the Horse," Frontiers in Veterinary Science, vol. 7, pp. 589-670, 2020.
14. M. R. Abess, M. K. Hussain, and Z. M. A. Jeddoa, "Lipid Profile and Coronary Heart Disease," Human Journals, vol. 11, no. 1, pp. 188-194, 2017.