Overview on Clinical Relevance of Antibodies Against Oxidized Low-Density Lipoprotein (oLAb) Within Three Decades by ELISA Technology

1. Introduction

Oxidative stress (OS) is defined as a disbalance in the redox cycle characterized by an excess of oxidants in relation to antioxidants, which disturbs the sensibly balanced equilibrium between these redox categories [1]. For many decades, numerous researchers have provided evidence that oxidative stress can be either caused by excessive consumption of pro-oxidants, excessive endogenous production of reactive oxygen species or the lack of antioxidants [2,3,4]. One of the most prominent consequences of OS is the attack of free radicals on polyunsaturated fatty acids (PUFAs), initiating a chain reaction that decomposes PUFAs to generate highly reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), colloquially termed “rancid lipids” in the course of the so-called “lipid peroxidation” (LPO) cascade, which was extensively studied in the 1990s exemplary on the PUFA and cholesterol-rich lipoprotein particle Low-Density Lipoprotein (LDL), among others in the lab of Hermann Esterbauer, who passed away in 1997 [5,6,7]. Cardio-vascular diseases and malignancies are the top two causes of death in developed countries. Both disorders are closely related to OS and LPO, which are partly responsible for initiating and exacerbating pathological processes, as postulated already by Brown and Goldstein in 1983 and demonstrated in numerous studies [8,9,10,11], see Table 1 for details.

The group of Steinberg, Witzthum, and Partharsarati [12,13] were the first to measure immunoglobulins reacting with aldehyde-modified proteins such as oxidatively modified LDL (oxLDL) in animal studies and to demonstrate that LPO occurs in vivo. Enzyme-linked immunosorbent assays (ELISA) coated with oxLDL were used to detect IgG antibodies in humans and several carnivorous and omnivorous animals [14,15]. Life-threatening conditions are associated with dramatic decreases of oLAb (oxLDL Antibody) titers in organisms, as could be shown in starved animals or humans suffering from septicemia, as well as significant decreases in non-surviving SARS-CoV-2 patients or during acute myocardial infarction. Physiologic antibodies, including oxLDL antibodies, are present in all mammalian species and are enormously important for the immune system to maintain homeostasis. These physiologic autoantibodies are produced in healthy individuals and perform protective and regulatory functions as a defence against diseases or for self-treatment after the onset of a disease. For instance, ageing erythrocytes show that immunoglobulin G (IgG) autoantibodies bind to senescent antigens on aged cells. Oxidation generates these neoantigens, which the immune system recognizes. As soon as the IgG is bound, the removal of the senescent cells is accelerated and carried out by macrophages [16]. Oxidatively modified LDL binds to the scavenger receptor on macrophages, which are converted to foam cells by uncontrolled intake as indicated in the scheme in Figure 1. Oxidized LDL expresses a large and, to date, unknown number of epitopes that induce the production of antibodies against these products [17]. Since the mid-1990s, Biomedica company has been a pioneer in producing the "oLAb" ELISA to determine IgG-antibodies against oxidized LDL based on the work of Tatzber and Esterbauer 1995 [18]. For comparability reasons, this review comprises exclusively studies performed with this commercial standardized method as works using custom-made assays are reviewed elsewhere [19]. With the help of this methodology, numerous studies have been conducted in recent decades, and findings regarding the immunological level of oxidative stress in various diseases have been made, which have benefited humankind and medicine [20,21,22]. In this context, Winklhofer-Roob et al. [23] mentioned the oLAb as fingerprints of an immune response to oxidized LDL. Another review stated that the assessment of oxLDL antibodies may more reliably reflect the level of oxidative stress than oxLDL [24]. The determination of the oLAb titer was very early proposed as a potential biomarker with clinical relevance very early, indicating the degree of in vivo oxidation [25].

Future aspects should be primarily concerned with the early diagnostic detection of excessive oxidative stress and initiating therapeutic intervention in preventative health care. Finally, an outlook on future aspects and possible applications of various antibodies against oxidized LDL, e.g., the application of human monoclonal antibodies against oxidatively modified lipoproteins may open new therapeutic concepts in the fight against life-threatening diseases like malignancies, septicemia, and cardiovascular diseases.

2. Antibody Titer Against Copper-Oxidized LDL in the General Population

At the beginning of antibody research against oxidized low-density lipoprotein (oxLDL) in the early 1990s, evidence accumulated that atherosclerotic disease is coupled with an elevated oxLDL antibody titer, and thus estimated as a health hazard [26]. Following a preliminary study by Tatzber et al. [27], who reported a normal range for the oLAb titer in 800 healthy men and women (20-60 years) between 400 and 600 mU/mL, which were determined in the course of a preventive medicine screening, Pincemail et al. [28] were the first who assessed and published the normal range for the antibody titer against oxidized LDL (oLAb) in the general population (n = 123) in an age range between 21 and 64 years. They determined a mean value of 468.37 ± 318.55 mU/mL with a normal range between 195 to 600 mU/mL. This standard range was substantiated in a large-scale study [29] with a random sample of 2793 participants (52.4% female) and an age range between 25-74 years. The median oLAb-titer was 300 mU/mL (5th-95th percentile: 74.4 – 1681) for males and 363 mU/mL (5th – 95th percentile: 80.4 – 1810) for females. This proportion was confirmed in a study with 73 healthy families, including their children in an age range between 8-22 years, with a slightly increased oLAb titer in women compared to men of the same age and a significantly higher oLAb titer in children compared to their parents [30].

The antibody titer in pregnant women (n = 40, age 27 ± 4.1 years) is not significantly different between the first and second trimesters (561.5 ± 424 vs. 581 ± 434 mU/mL) [20]. Nevertheless, in a comparative study of normotensive pregnant women (n = 49) with 26 women affected by pregnancy-induced hypertension, a significantly decreased oLAb titer associated with hypertension (348 ± 388 mU/mL, v.s. 579 ± 400 mU/mL, p < 0.01) was observed. This is of interest for neonates, as their oLAb titer is similar to their mother´s post-partum titer due to transplacental transport. Delivery is associated with oxidative stress for both mothers and newborns, as indicated by significantly increased levels of the LPO-product MDA versus controls and even higher concentrations in newborns accompanied by decreased abundance of the antioxidant enzyme superoxide dismutase (SOD). Consequently, the oLAb titer doubles within the first three months in newborns, while antioxidative glutathione (GSH) levels decrease significantly. Furthermore, lipid parameters were significantly increased after three months [31]. This working group of Steinerova et al. proved that the antibody titer depends on nutrition in newborns after three months, although there is an initial oLAb correlation with their mothers (r = 0.79; p < 0.001). In detail, breast-fed babies showed an average oLAb titer of less than 100 mU/mL, while formula-fed babies showed an oLAb titer of about 4000 mU/mL. This discrepancy could be due to a reaction to foreign proteins from cow's milk or modified molecules, especially since increased DNA damage was identified in formula-fed infants associated with oxidized pyrimidines. They identified a bovine milk protein, i.e., beta-casein A¹, to be atherogenic; thus, almost exclusively male formula-fed infants produced high levels of oLAb in consequence [32,33]. However, as an Italian research group has shown, the diet-dependent influence on the oLAb titer is not limited to newborns. They conducted a prospective population-based longitudinal investigation, including 101 males and 164 females, pharmacologically untreated non-smoker subjects, which were evaluated for factors that might affect their oLAb titers. Although they did not observe significant effects of the dietary approach, which did not influence the oLAb titer in a clinically relevant manner, they observed a significant predictor of log-ox-LDL antibody level in a multivariate analysis for age (HR -0.19; 95%CI -0.115 - -0.104, p = 0.001; adjusted r² = 0.581). In males, the log-ox-LDL antibody level was only predicted by the current dietary animal lipids content (HR -0.12; 95%CI -0.127 - -0.109, p < 0.001; adjusted r² = 0.128) in comparison to females, where age (HR -0.11, 95%CI -0.119 - -0.107, p < 0.001), soluble fibers (HR 0.11, 95%CI 0.104 – 0.110, p < 0.001) and alcohol intake (HR 0.18; 95%CI 0.102 – 0.114, p < 0.013, adjusted r² = 0.182) were the best predictors of oLAb levels [34]. Antibodies against oxidized LDL were not significantly different between alcoholic patients with slightly affected liver function but without any disease (n = 35, age 42 ± 9y) compared to healthy blood donors (n = 60, age 40 ± 8y) [35]. Use of oral contraception in 40 to 48-year-old females was associated with a significant prooxidative effect through increased total peroxides compared to non-contraception users or intrauterine device users. Elevated copper concentrations decreased ß-carotene and γ-tocopherol without affecting the oLAb titer. The strong correlation between copper and total peroxides increase indicates lipid peroxidation enhancement in females using oral contraception. These authors conclude with the comment to monitor these parameters and suggest antioxidant supplementation considering the potential risk for venous thromboembolism and cardiovascular disorders [36]. In the Wandsworth Heart and Stroke Study population, different healthy ethnic groups, i.e., 250 Whites (113 females), 169 African origin (91 females) and 196 South Asian (92 females) were determined for soluble serum oLAb levels [37]. The oLAb titer was significantly increased in subjects of African origin and South Asian origin. The titer was negatively associated with triglycerides and positive with s-VCAM-1, especially in female South Asian individuals, with a decrease in Vitamin C level and an increase in plasma homocysteine concentrations, which indicates a condition of increased risk for coronary heart disease (CHD) and cardiovascular disease (CVD).

The ethnic influence on the oLAb titer is also evident from a study of 158 women and 158 men in Japan [38] with relatively low oLAb mean values (female 209 (152.6 – 312.5); male 170.7 (130.9 – 301.2)) compared to the standard European normal range [28]. A large-scale cross-sectional study from Poland, including 3154 individuals aged 65 and older, showed that advancing age was significantly associated with decreased paraoxonase 1 (PON1) activity and increased oLAb. The age-dependent diminished activity of PON1 was related to inflammation and the generation of oLAb as a counterbalance to elevated oxidative stress. In detail, the authors observed a significantly lower oLAb titer in males than females and an inverse relation to triglycerides. They concluded that both PON1 and oLAb act independently and synergistically against the amount of oxLDL as PON1 activity decreases with age. Thus, oLAb becomes more critical in eliminating oxLDL with extraordinarily high titers in the oldest group [39].

2.1. oLAb and the Innate Immune System

The oxidation of LDL through ROS, including hydroxyl radical (OH^∙), superoxide anion (O₂^∙−) or hydrogen peroxide (H₂O₂) leads to the formation of modified phospholipids with a surface structure similar to that of damaged cells, which are recognized by antibodies against oxidized LDL and scavenger receptors, which also play an essential role in the recognition and clearance of bacteria and apoptotic cells [12], sketched in the scheme below.

Figure 1. Left part: Oxidative stress gives rise to free radicals, which initiate and promote the decomposition of polyunsaturated fatty acids (PUFA) in the cell membrane´s or LDL phospholipid Sn2-position to produce highly reactive aldehydes such as MDA and 4-HNE, which react with proteins in close vicinity such as ApoB on LDL. Damaged proteins are phagocytosed by macrophages, which process them in lysosomes into smaller peptides, which are then presented as antigens to immune cells, and B-cells produce IgG antibodies recognizing oxidatively modified proteins, such as ox-LDL. Right part: In the circulation, oxLDL is recognized by oLAb and cleared from circulation via FcR (Fc-Receptor) on macrophages. Continuous uptake of oxLDL by macrophages gives rise to foam cell formation.

Figure 1. Left part: Oxidative stress gives rise to free radicals, which initiate and promote the decomposition of polyunsaturated fatty acids (PUFA) in the cell membrane´s or LDL phospholipid Sn2-position to produce highly reactive aldehydes such as MDA and 4-HNE, which react with proteins in close vicinity such as ApoB on LDL. Damaged proteins are phagocytosed by macrophages, which process them in lysosomes into smaller peptides, which are then presented as antigens to immune cells, and B-cells produce IgG antibodies recognizing oxidatively modified proteins, such as ox-LDL. Right part: In the circulation, oxLDL is recognized by oLAb and cleared from circulation via FcR (Fc-Receptor) on macrophages. Continuous uptake of oxLDL by macrophages gives rise to foam cell formation.

In this respect, it should be noted that pneumococcal vaccination is associated with an increase in oLAb, indicating a mechanistic link between vaccination and a protective effect on cardiovascular disease. After multivariate adjustment for potential confounders, this association remained significant (p = 0.04). This seems particularly important for older people, and pneumococcal vaccination is now recommended for these subjects as their immune systems weaken with age, and they are at an increased risk for vascular diseases [40].

2.2. Smokers

Heavy smokers (>20 cigarettes/day) showed a significant increase in the oxidative stress DNA marker 8-hydroxy-deoxyguanosine (8-OHdG), associated with a significant decrease in α-carotene, β-carotene, β-cryptoxanthin and zeaxanthin. In males, a significant negative correlation was shown between oLAb and β -carotene (r = 0.33, p<0.01) with the number of cigarettes per day. Thus, the authors recommended 8-OHdG, oLAb and carotenoids as valuable biomarkers to evaluate the oxidative conditions caused by smoking [41]. In an ethnic study [37], it was found that in smokers, the oLAb titer was significantly decreased (384 U/L, 95%CI 316-468 U/L) compared to non-smokers (430 U/L, 95%CI 471 – 596; p<0.009). In stenotic patients, an increased oLAb titer was observed in smokers (472.2 ± 65.9 mU/mL) compared to non-smokers (275.6 ± 33.5 mU/mL). Increased oLAb titer were significantly pronounced in unstable angina (p < 0.005) [42].

2.3. Antioxidants

The benefits of polyphenols in olive oil were established in a clinical trial [43] as they promote an increase of oLAb titers, with the most pronounced effect in subjects with high oxidized low-density lipoprotein (ox-LDL) concentrations. The oLAb titer increased with the polyphenol content in a dose-dependent manner and was inversely associated with oxLDL. This was substantiated by the correlation of the oLAb titer with the amounts of polyphenols bound to LDL cholesterol. Another comparative study between Iranians and Austrians [44] showed that Iranians indicated a higher oLAb titer, lower cholesterol levels, LDL and HDL but increased levels of lycopene, canthaxanthin and lutein. An intriguing perception in this study was the significantly increased oLAb titer (p < 0.01) accompanied by an increased resistance of LDL (lag phase; p < 0.005) with a concomitant decrease of MDA levels after combined daily intake of beta-carotene (30mg) and alpha-tocopherol (400IU) over ten weeks. Based on these studies, the intake of fat-soluble antioxidants appears to increase the oLAb titer. On the other hand, pomegranate juice (200mL per day for six weeks), which is abundant in polyphenols, prevented oxidative stress in type 2 diabetic patients, reduced the oLAb titer and increased the total antioxidant capacity and PON1 activity [45]. In a cross-sectional study with 551 community-dwelling older adults between 65 to 94 years from Dicomano, Italy, it was shown that wine consumption, through its antioxidant properties affects oLAb titers in older populations [46]. Oxidative stress was significantly reduced during a 24-week therapy by Rosuvastatin, including an immunomodulatory effect. This polyphenol attenuates oxidative stress through a decrease of total peroxides and antibodies against oxidized LDL besides cholesterol lowering, thus supporting the prevention of atherosclerotic diseases [47]. Young patients with bipolar disorder exhibited an inverse association between vitamin D and the oLAb titer (r = -0.34; p < 0.05) [48].

2.4. Body Mass Index (BMI)

Body mass index is associated with increased insulin resistance and hyperglycemia in elderly men, especially in “young-old men (62-74 years)” with a significantly increased BMI and decreased oLAb compared to “old-old men (75-83 years)” [49]. Antioxidants and thiobarbituric acid reactive substances TBARS were not significantly different between these groups, indicating no age-dependent difference concerning antioxidants between young and older men except for lycopene [50]. The inverse relationship between BMI and oLAb was confirmed in a cohort of 2190 subjects (1283 females), showing a significant oLAb decrease with the lowest titer at BMI >30. This correlated with the highest cholesterol, LDL, uric acid and peroxide concentrations, while bilirubin and HDL, were lowest in this BMI group. A linear increase in the antioxidant status (TAS) occurred up to a BMI of 34.9, presumably due to the steady increase in uric acid, an excellent antioxidant correlating significantly with the TAS. In those subjects with a BMI of 35 and above, despite a further increase in uric acid, there was a decrease in TAS coupled with a doubling of peroxides [51]. This crucial situation is associated with the upregulation of oLAb in that high peroxide values tend to lead to increased oLAb titers [52]. In a randomized-controlled study, the effect of Lactobacillus casei Shirota was monitored for 12 weeks in 30 subjects with metabolic syndrome. Apart from a significant reduction in the soluble vascular cell adhesion molecule-1 level (p = 0.008), no other significant differences were observed, neither in insulin sensitivity, β-cell function, endothelial function nor for inflammation markers or oLAb (baseline 563 mU/mL vs. 12 weeks 529 mU/mL) [53].

In a study with anorexia nervosa (AN) patients, the lowest oLAb titres were measured in the lower subcutaneous adipose tissue (SAT) group (BMI = 14.5 ± 1.1) [54]. Although the antioxidative and oxidative stress biomarkers were below reference values in the higher SAT group (BMI = 16.1 ± 0.9), these parameters were still better than those in the lower SAT group. This is crucial information, especially since a previous study in subjects generally defined by a body mass index (BMI) <18.5 showed no significant differences in oLAb levels compared to normal weight subjects [51]. Therefore, conducting a detailed oxidative stress analysis for AN patients is essential and reasonable, as this group with lower SAT is associated to an extraordinary degree with increased oxidative stress, which also impacts the therapeutic outcome and long-term recovery.

2.5. Exhaustive Exercise—Sports

Exhaustive exercise is associated with increased oxygen consumption and metabolism. Thus, reactive oxygen species (ROS) become elevated during training and competition, which induces the generation of oLAb to protect against oxLDL damage and further consequences against atherosclerotic modifications. Half of soccer and basketball players indicate very high titers of oLAb—up to 6000 mU/mL. This was accompanied by a trend towards lower vitamin C levels in athletes with high oLAb titres (8.49 ± 3.14 µg/ml vs 10.39 ± 2.55 µg/ml), apart from the fact that it was not significant [28]. In another study, the majority of soccer players (n = 7) were indicated with a mean oLAb titer of 1102.8 ± 45.8 mU/mL and four players with 31.5 ± 12.6 mU/mL, although antioxidant levels were in the normal range and did not differ between groups [55]. Nansseu et al. [56] reported a significant increase of the oLAb titer during the competition season as indicated at three-time points (median oLAb titer 653 mU/mL March; 777 mU/mL May; 1037 mU/mL July; p = 0.006) in eighteen professional soccer players. They further observed a concomitant decrease in uric acid and total antioxidant capacity.

In professional American football players, likewise in half of the athletes, a significant increase in total peroxides and oLAb was shown in the middle of the competition season, which levelled off at the end. The oxidative stress was neither predictable from baseline values nor on the status of individual antioxidants [4]. The oxidative stress in the course of a competition season (July - January) in the Austrian Men's Alpine Ski Team showed a high load in December, whereby the basic oLAb titer (1036 ± 328 mU/mL July) was decreased to 439 ± 150 mU/mL, which indicates a significant consumption of these antibodies. The athletes recovered to near pre-season levels during the days off over Christmas and New Year. The oxidative stress parameters also correlated with the athletes' performance, with the successful athletes having only about 50 per cent of the peroxide concentrations and an oLAb titer at least twice as high as the low-performing skiers. In addition, the latter were also susceptible to infections in December [57]. In this context, it should be noted that the antioxidant concentrations were, on average, all within the normal range (α-tocopherol, γ-tocopherol, ascorbate serum concentrations) or, in the case of β-carotene even twice as high as the reference values. The oLAb titer of active cyclists decreased significantly compared to that of the control group, and the titer of ex-cyclists was between these two groups, although not significantly different from that of the control group. The training effect remained for the improved antioxidant status for one year, while the effect on oLAb was lost as soon as one became sedentary. Interestingly, there was no correlation between training hours and the oLAb-titer [58].

Regarding non-exhausting conditions in a group of 41 postmenopausal female members (age 59-71 years) who completed a training program over eight weeks (3 times a week) on an ergometer, the oLAb proved to be a robust parameter that remained constant under these health-promoting conditions, primarily as the anthropometric parameters such as BMI or body weight also remained unchanged. However, the positive effect of physical activity had an impact on glucose levels, LDL-cholesterol and antioxidant capacity [59].

2.6. Hyperlipidemia

A low-grade inflammatory state, metabolic disturbances in the lipid profile, and insulin resistance occur in Familial Combined Hyperlipidemia (FCH). It is associated with an increased oLAb titer even though the oLAb-titer of the control group was in the lowest section of the normal range [60]. In a small number (5 males and 1 female in the range of 41-60 years) of heterozygous familial hypercholesterolemia subjects, a median oLAb titer of 269 mU/mL was observed before and 315 mU/mL after LDL apheresis, which was not significantly different. This form of treatment did not change the level of 8-iso-PGF2α but increased the lag time and decreased the concentration of TBARS [61].

2.7. Diabetes Mellitus

In the case of insulin-dependent diabetes mellitus patients, the severity of the disease was inversely related to the oLAb titer, indicating the requirement of these antibodies to react with the antigen, as oxLDL immune complexes were identified in those patients with longer diabetes duration and higher actual and mean HbA1c levels [62]. Childhood type 1 diabetes mellitus (T1DM) is associated with significantly higher oLAb titers than controls, which indicates an inverse correlation between oLAb and HbA1c levels. This is an immunological defense against an excess of oxidative stress. In detail, those subjects with reasonably good metabolic control (n = 21), i.e., HbA1c levels less or equal to 9%, have shown a marked increase in oLAb (488 mU/mL). In contrast, the poor metabolic control (n =15) showed a markedly decreased antibody titer (183 mU/mL), most probably due to the worse redox balance by forming more antigen-antibody complexes reflected by a lower titer of free antibodies [21]. In young type 1 diabetic patients, oLAb titer decreased with age and duration of diabetes, which fits atherosclerotic processes as oLAb is an immunologic expression of lipid peroxidation. In this respect, the inverse correlation with vitamin E can also be classified, with the lipid-soluble antioxidant representing a significant factor in protecting against the in vivo oxidation of LDL. On the other hand, there were no significant differences in oLAb titers in these young diabetics, who were generally under reasonable diabetic control, regardless of whether they had no complications, subclinical retinopathy, neuropathy or nephropathy [63].

3. oLAb and Cardio-Vascular Diseases

3.1. Systemic Sclerosis

In patients suffering from Systemic Sclerosis (SSc), the oLAb titer increased predominantly in the early phase in both the limited and diffuse SSc (428.9 ± 417.1 mU/mL), where the immune response to lipid peroxidation products is much stronger than in the advanced phase (302.7 ± 89.9 mU/mL) of the disease. Nevertheless, the pathophysiology of the limited form is predominant and associated with extended reperfusion injury compared to the diffuse SSc, where the episodes are significantly fewer due to the loss of capillaries [64].

3.1.1. Peripheral Arterial Disease (PAD)

In twenty-one diagnosed patients by peripheric angiography suffering from peripheral atherosclerotic disease (PAD), the oLAb titer was increased (357.44 ± 177.71 mU/mL) compared to the control group (266.35 ± 113.19 mU/mL; p < 0.05). Moreover, in patients with severe Fontaine claudication (IIb), the oLAb titers were higher (403.03 ± 223.67 mU/mL) compared to patients with mild Fontaine claudication (IIa) (316.00 ± 119.02 mU/mL). As a result of increased oxidized LDL in these patients, the increased oLAb titer is of great importance due to its physiological function of removing oxLDL from the circulation and the artery wall [65]. Vascular walls are damaged during percutaneous transluminal angioplasty (PTA), which is finally associated with ischemia/reperfusion injury. This technique is primarily performed in patients with Fountaine stage IIb and IV. The latter group is characterized by significantly increased peroxide concentrations, while revascularization is associated with a significant decrease in oLAb titers, which is why this antibody could be important as a revascularization marker [66,67].

3.1.2. Coronary Balloon Angioplasty

The prognostic value for oLAb was investigated in patients undergoing primary coronary balloon angioplasty for acute ST-elevation myocardial infarction (STEMI). Restenosis was observed in approximately one-third of patients. In this group, the oLAb titers were significantly decreased before angioplasty, while oxidized LDL did not differ between groups. Therefore, a sufficient titer of oLAb is a prerequisite for clearance of ox-LDL from circulation to avoid restenosis after primary angioplasty [9].

3.1.3. Cardio-Vascular Disease (CVD) and Intima Media Thickness (IMT)

In healthy subjects, a significant inverse association between oLAb titers and the intima-media thickness of the carotid arteries, even after multiple regression analysis, indicated the immune response as a safeguard against oxidized LDL and its detrimental health implications, such as at an early stage of atherosclerosis [68].

In cardiovascular disease (CVD) patients an increased ox-LDL was associated with decreased oLAb titers. Intima-media thickness (IMT) values were higher in uremic patients versus controls and in CVD patients compared to patients without CVD which correlated positively with the ox-LDL/oLAb ratio in comparison to an inverse association to oLAb. The authors suggested the ox-LDL/oLAb ratio as a new predictor of IMT, with an important role in distinguishing between patients with and without cardiovascular complications [69]. The inverse association between oxLDL and oLAb was also reported in male subjects with stable coronary heart disease (CHD). A significant increase in ox-LDL was associated with the consumption of oLAb and an increase in Superoxide-Dismutase (SOD) and glutathione-peroxidase activity (GSH-Px) [70]. In a clinically healthy population, plasma ox-LDL concentrations were also negatively correlated to oLAb regarding the intima-media thickness of the common carotid arteries. Those subjects with high oLAb and low ox-LDL had the most negligible intima-media thickness of the common carotid arteries [71]. The concept that oLAb, i.e., the immunity against oxidized low-density lipoprotein, plays an anti-atherogenic role was supported by the significant inverse relation between oLAb titer and intima-media thickness of femoral arteries (FA-IMT) and the inverse trend between oLAb and intima-media thickness of carotid arteries (CA-IMT) in end-stage renal disease patients [72]. The greyscale median of the intima-media complex (IM-GSM), a tool for atherosclerosis detection alongside the intima-media thickness, was associated with several inflammatory markers and oxidative stress biomarkers, among others with an inverse relationship to oLAb in an elderly population with 1016 participants from the community of Uppsala, Sweden [73]. In CAD patients with no vessel disease, the oLAb titer was significantly increased compared to controls. This finding indicates an advanced generation of these antibodies as a countermeasure upon an increased incidence of antigen. In advanced stages of the disease, i.e., with single-, double- or triple-vessel disease, these antibodies are consumed, which could be determined by decreased titers [74].

3.1.4. Angina Pectoris

A study of patients diagnosed with angina pectoris showed an increased oLAb titer of 27% compared to the control group [75].

3.1.5. Acute Myocardial Infarction (AMI)

The dynamic of oLAb titers is probably best pictured in acute pathological settings such as AMI, and it is thus not surprising that seemingly contradictory results were reported by several research groups. Stable acute myocardial infarction (AMI) patients without classical risk factors were identified with very low oLAb titers, significantly differing from population controls indicating an oLAb cut-off value for AMI risk at 165 U/L applying a bootstrap method with 1000 replications. Patients with an oLAb titer of 165 U/L or less had a higher risk of AMI with a crude odds ratio (OR) of 6.15 (95% confidence interval (CI): 2.24-16.9) and after adjusting by the Framingham-risk-adapted score and ox-LDL, the natural logarithmic level of oLAb maintained the independent association (OR of 0.43, 95% CI: 0.23-0.79). On the other hand, the ox-LDL level between controls and cases did not differ [76]. The working group of Gruzdeva et al. reported that the oLAb titer in myocardial infarction patients (MI) was significantly increased compared to controls. In addition, complicated MI (Killip class II-IV) had higher oLAb titers on day one compared to non-complicated MI (Killip class I), which kept high until day twelve [77]. The same group performed a study including 400 patients with myocardial infarction who were assigned to ST-elevation, which were divided into three groups, i.e., A) one vessel ≧ 75% stenosis, B) two vessels ≧ 75% stenosis and C) three or more coronary arteries with ≧ 75% stenosis and blood samples were drawn at the first and 12^th day. Besides increased lipid values such as free fatty acids, total cholesterol, LDL, triglycerides, peroxides and oxidized LDL, significantly increased antibody titers against oxLDL were also determined compared to the control group. In group A, the titer was increased by 37% (p < 0.01), in group B by 69% (p < 0.005) and in group C by 134% (p < 0.004) on the first day compared to the control. The antibody titer also increased with the number of affected arteries, i.e., by 17% in group B and 70% in group C compared to group A [78]. The authors concluded that FFA or antibodies against oxidized LDL would be the most informative indices for the severity of atherosclerotic lesions, which are elevated in multivessel disease during hospitalization.

On the contrary, the study by Nikolic-Heitzler et al. [79] confirmed the results of Schumacher et al. [14] regarding the initial consumption of oLAb after percutaneous coronary intervention due to acute myocardial infarction: a significant decrease after three days and a significant increase after another four days. In this respect, the measurement time points appear to be decisive since Schumacher et al. [14] monitored a period from admission to 48 hours afterwards, Nikolic-Heitzler et al. [79] from admission via 2 and 4 hours as well as after 3 and 7 days, in contrast to Gruzdeva et al. [77], who analyzed only at two-time points, i.e., after one and twelve days. An antibody titer increase was already reported after seven days [79].

3.1.6. Stroke

The antibody-titer against oxidized LDL was significantly decreased in stroke patients compared to controls, especially pronounced in hypertensive patients [80].

4. Renal Disease

In renal disease patients oLAb titers were increased both in patients after kidney transplantation and more pronounced in hemodialysis patients, presumably due to the atherogenic lipids and lipoprotein ratios [81]. In hemodialysis patients, superior to patients with cardiovascular disease, an increase of the vascular endothelial growth factor was associated and positively correlated with the Cu/Zn superoxide dismutase level (p < 0.01) and the oLAb-titer (p < 0.05). This indicates a local tissue production of ROS and VEGF following the hypothesis that the global activation of macrophages and smooth muscle cells, which synthesize and secrete these molecules, are responsible for their accumulation in plaques. This was supported by a multivariate analysis showing that Cu/Zn SOD and oLAb are strong independent variables linked to VEGF [82]. Predialysis patients with carotid plaques had significantly increased oLAb titers compared to those without plaques, as confirmed by Rubba et al. [83], who found an independent correlation between the plaques in the common carotid artery and the oLAb—but not in the bifurcation. An inverse correlation between oLAb and vitamin E indicates the protective effect of tocopherol against the oxidation of LDL, with malnourished chronic renal failure patients showing significantly lower vitamin E levels [84].

In the case of intermittent hemodialysis (IHD), inflammatory parameters are affected by renal replacement therapy that was associated with an increase of total peroxides, in contrast to procalcitonin, malondialdehyde and oLAb, which were unaffected [85]. In patients with chronic renal failure elevated VEGF levels were reported in association with an increase in inflammation, total peroxides and Cu/Zn SOD levels while oLAb was not significantly different. Almost all plasma levels of oxidative stress markers, inflammatory proteins and vascular endothelial growth factor (VEGF) were increased at chronic renal failure stage 3 to 5, indicating an early appearance of oxidative stress during the progression of renal insufficiency [86]. A decreased oLAb titer was observed in children on hemodialysis. Especially those patients with very low oLAb-titers (<137 mU/mL) indicated an increased hs-CRP level, and lower levels of albumin, apolipoprotein A-1 and high-density lipoprotein. The situation was even worse in patients with dyslipidemia [22]. Differentiation in hemodialysis patients with or without vascular calcification (VC) in the legs could neither be detected by oLAb (416.9 ± 287.1 mU/mL -VC vs 341.5 ± 307.3 mU/mL +VC, p = 0.131) nor by the numerous biomarkers tested, apart from a decreased concentration of HDL. However, an inverse correlation between oxLDL and oLAb was found in this group of hemodialysis patients [87]. The oLAb titers in kidney graft recipients are significantly lower compared to controls. In the post-transplant period, these titers decreased further, most likely due to permanent immune-suppressive treatment, within the first six months and remained at this diminished level until the observation period of 24 months. Low pre-transplant oLAb titers (207 [89-855 U/L]) were associated with early graft loss on account of acute rejection versus patients with graft survival longer than three months (546 [79-16748 U/L]) besides a higher frequency of ischemic heart disease (p < 0.05) and an increased chronic graft damage score of greater than 6. The authors highlight the protective effect of oLAb, which binds oxidatively modified lipids and thus protects the vessel walls from damage, as they were able to substantiate that a low oLAb titer is associated with an increased risk for complications in kidney graft recipients [88]. Another study with 71 post-renal transplant patients confirmed an identical oLAb titer (225 mU/mL; range: 40-850 mU/mL), which was significantly lower compared to hemodialysis patients (n = 33; 462 mU/mL; range: 89-850 mU/mL, p < 0.01) but higher than those of the control group (n = 89; 175 mU/mL; range: 45-350 mU/mL, p < 0.05) with the restriction that the oLAb titers of the control group were below the normal range of 200-600 mU/mL [81].

5. Cancer

There was no association between oLAb levels and the risk of colon cancer, while elevated levels of oxLDL were associated with an increased risk of colon cancer, independent of confounding factors [89]. In colon cancer patients, an association with advanced age and increased oLAb titers, especially at the beginning of the disease at stages 1 and 2, depending on the localization, were reported. In contrast, oLAb titers were significantly lowered in advanced stages, i.e., in severe cases. The authors described this effect as an individual difference in the host's immune response to cancer [11]. Increased oxidative stress was also demonstrated in patients with hepatocellular carcinoma, whose oLAb titers were bisected compared to the control group [90].

6. SIRS and Sepsis

In intensive care unit patients, there was a significant increase in oLAb titers accompanied by decreasing inflammation parameters in contrast to non-survivors, which indicated an inverse sequence of events [91].

7. Thyroid

Hypothyroidism is associated with an increased production of antibodies against oxLDL, compared to a healthy control group. On the other hand, a decreased oLAb titer was observed in hyperthyroid patients. Thus, thyroid dysfunction leads to oxidative stress, lipid peroxidation, and atherosclerosis, which was indicated by a decrease in TAC and an increase in total peroxides. Therefore, especially in hypothyroidism, the “response to injury” stimulates the immune system [92].

8. Multisystem Disorder—Behçet’s Disease

Patients suffering from Behcet’s disease, a multisystem disorder with relapsing inflammatory processes and its predominant histopathology of vasculitis, are prone to oxidative stress. ROS overstrain the antioxidant system with a significant decrease of antioxidant enzyme activities such as catalase and superoxide dismutase as well as glutathione, total antioxidant status and shortened lag-time. As a counteraction to elevated lipid hydroperoxides, the oLAb-titer increases as an immunological stress response [93,94].

9. Congenital Disorder—Cystic Fibrosis

In cystic fibrosis patients, oxidative stress was indicated through a decrease of retinol, α-tocopherol and β-carotene and a significantly increased and exorbitant oLAb as well as TBARS. The authors concluded that an increased antibody titer against oxidized LDL might be an indication of an imbalance in the oxidant-antioxidant system, with an excess of oxidative stress in the presence of chronic lung inflammation in CF [95].

10. Chronically Inflammatory Diseases

10.1. Morbus Crohn

In Crohn’s disease patients oLAb titers better reflected the disease severity than oxLDL and are positively correlated to the proinflammatory interleukin 1 (IL-1) (p = 0.038) and disease activity index (p = 0.035) [96].

10.2. Psoriasis

In psoriasis patients, who suffer from a chronic and recurrent inflammatory skin disease, there is an increased atherogenic tendency due to a disturbance of the oxidant-antioxidant balance through a decrease of endogenous antioxidant enzymes, including the total antioxidant status in contrast to oxidative stress markers. E.g., malondialdehyde and lipid hydroperoxides were increased, as well as the antibody titer against oxidized LDL [97]. In this chronic inflammatory skin disease, which is associated, among others, with dyslipoproteinemia and, in further consequence, with an increased incidence of cardiovascular disease, the oLAb titer was significantly increased in patients compared to healthy controls. The authors concluded that an imbalance of the oxidant-antioxidant system was responsible for an increased inflammation even though LDL-C levels of patients and controls did not differ significantly. The oLAb titer correlated significantly with the Psoriatic Area and Severity Index (PASI) score, indicating the clinical severity of the disease and concomitant inflammation due to elevated PMN elastase and alpha-1 antitrypsin levels [98].

10.3. Vaginitis

In 45 patients with recurrent thrush vaginitis, increased total peroxides (399 µmol) and oLAb titers (648 mU/mL) were reported with a corresponding drop in TAC. Through an antioxidant therapy, 77% of the patients became symptom- or recurrence-free [99].

11. Allograft Transplantation

In liver and heart allograft transplantation patients, an inverse correlation was observed between malondialdehyde and oLAb (r = 0.61, p<0.001 in the case of liver and p = 0.54, p<0.05 in heart transplantations), indicating oLAb as a second barrier against lipid peroxidation [100,101].

12. Post-Traumatic Changes

The lowest oLAb titers in patients with bone fractures and/or traumatic brain injury (TBI) were found in the first week. However, those with TBI had significantly higher titers than those with isolated fractures. Over four weeks, there was a significant gradual increase in all patients. However, an increase in the oLAb titer compared to controls was only significant in TBI patients in the third and fourth weeks [102].

13. COVID-19

After the infection with SARS-CoV-2, the oLAb titer significantly decreased in recovered and diseased COVID-19 patients, i.e., with an enhanced decrease in non-survivors, albeit not significantly different from survivors [103]. It should be mentioned that there was a persistent inverse correlation between the concentration of 4-hydroxynonenal and oLAb in the blood of non-survivors, compared to a positive correlation in survivors at the end of the observation period [104]. This effect was assigned to inflammation and the attack of free radicals, which was associated with alterations in lipid metabolism, thus resulting in systemic oxidative and vascular stress.

14. Conclusions

Over three decades, numerous clinical studies have determined the antibodies against oxidized LDL and established correlations with clinical symptoms and diverse other biomarkers. The oLAb titer in the general population is heterogeneous, while the titers within an individual are relatively constant. In this respect, the measurement of the oLAb titer is solicited as a robust biomarker, e.g., in annual health examinations. These physiologic autoantibodies serve to dispose of oxidatively modified molecules and cells, whereby binding to the antigen is accompanied by a reduction in the titer. This immunological support is a second barrier and is particularly useful in cases of increased oxidative stress, e.g., in the case of intensive care patients or myocardial infarction patients. The oLAb is convenient for monitoring the course of these severe illnesses to monitor progress in recovery. Monitoring these antibodies is further indicated in heavy workers and elite sports, where athletes are exposed to increased oxidative stress due to heavy physical exhaustion, which is also associated with performance. Future research should also focus on antioxidants and their correlation to oLAb, especially as there is clear evidence that antioxidant supplementation can significantly influence antibody titers. In this regard, it should be noted that oxidative stress takes place at different levels, and therefore, additional OS biomarkers and the knowledge of the antioxidant capacity are necessary for a profound assessment. However, antibody research is non-exclusive beneficial in diagnostics but could also significantly contribute therapeutically to the fight against oxidative modifications in the future through monoclonal antibodies.