In a 2nd step an AAR value of 1.67 was calculated by receiver-operating curve analysis as an optimal cut-off value to discriminate between CLI and non-CLI with a sensitivity of 34.1% and a specificity of 81.0%. Consequently, we categorized our cohort into 2 groups: 1st group with an AAR < 1.67 containing 1385 patients and a 2nd group with an AAR > 1.67 containing 397 patients. The 1st group contained 329 (23.8%) CLI patients whereas the 2nd group included 166 (41.9%) patients with CLI. The difference between groups was statistically significant (P < 0.001). Between the 2 AAR groups we found further statistically significant differences in other vascular endpoints (prior myocardial infarction 54 [3.9%] vs 28 [7.1%], P = 0.01) and in inflammatory parameters (C-reactive protein [CRP] [median 4.3 mg/L (2.0–11.5) vs 8.1 mg/L (2.9–28.23)] and fibrinogen [median 388.0 mg/dL (327.0–493.0) vs 427.5 mg/dL (344.25–530.0)]; both P < 0.001) as well (Table 2). We also did statistical analyses on the correlation of AAR with CRP and fibrinogen. We calculated a Pearson correlation and a Spearman rho as well for AAR and CRP as for AAR and fibrinogen. For both parameters, we could confirm a statistically significant but not relevant correlation between the AAR and CRP/fibrinogen (P-value of <0.001; for AAR and CRP: Pearson r = 0.18; Spearman rho = 0.14; for AAR and fibrinogen: Pearson r = 0.08; Spearman rho = 0.09).
In a 3rd step AAR > 1.67 was used as a variable in a binary logistic regression model to evaluate this value as an independent risk factor for CLI. In this model AAR > 1.67, sex, type 2 diabetes, age > 75 years, coexistence of congestive heart failure, arterial hypertension, CRP, and renal impairment were included. Type 2 diabetes, age >75 years, and renal impairment were included as these variables showed a close association with a coexisting CLI in studies published recently from our group[13,14] and CRP was included as this is an established parameter reflecting vascular inflammation. Even after adjustment for these parameters AAR > 1.67 was associated with an OR of 2.0 (95%confidence interval 1.7–2.3, P < 0.001) for CLI (Table 3).
In this study, we were able to demonstrate that an AAR > 1.67 is associated with CLI in PAOD patients. Even after adjustment for other main CLI risk factors like renal impairment, diabetes, and age >75 years, AAR > 1.67 was associated with a 2-fold increase in CLI risk. However, not only CLI was more frequently found in the high AAR group. Endpoints due to atherosclerotic lesions in other vascular beds, like myocardial infarction, were also more frequently encountered in this group. Even entities associated with coronary artery disease, like congestive heart failure and atrial fibrillation, were significantly more prevalent in the group with AAR > 1.67.
NAFLD might be one reason for our findings concerning elevated AAR and association with cardiovascular endpoints in our study. This entity was only recently evaluated in over 3000 patients included in the Framingham Heart Study. The authors were able to show that NAFLD was significantly associated with subclinical cardiovascular outcomes defined as coronary artery calcium and abdominal artery calcium. The mechanisms by which hepatic steatosis might contribute to vascular disease is still under discussion. Of course both entities share various risk factors such as diabetes, elevated triglyzerides, and hypertension. However, Mellinger et al were able to show that the association of NAFLD with subclinical atherosclerosis was independent of these established risk factors. The authors discuss that microRNAs might play a certain role as have been shown to be released by the liver and promote vascular disease.
The close correlation of NAFLD with abdominal obesity and insulin resistance makes it difficult to distinguish the causal relationship underlying the increased risk of cardiovascular disease in these patients. Hepatic steatosis is associated with increased production of interleukin-6 and other proinflammatory cytokines by hepatozytes and nonperynchymal cells, including Kupfer cells.[19–22] These increased cytokine expression is likely to play a key role in the progression of NAFLD and cardiovascular disease as well. Several case–control studies were able to show that inflammatory markers also reflecting inflammation in atherosclerosis patients, like CRP, interleukin-6, and fibrinogen were highest in NAFLD patients, intermediate in patients with simple steatosis and lowest in control subjects without steatosis. These differences were independent of obesity and other potentially confounding factors. Increased CRP promotes inflammation and atherosclerosis via increase in expression of plasminogen activator inhibitor-1 and adhesion molecules in endothelial cells and leading to an increase in LDL uptake into macrophages.
Of course AAR is influenced by various environmental and physiological parameters. First alcohol intake is a common cause of elevated liver enzymes. As patients with relevant alcohol intake were excluded from our cohort, this influence seems to be negligible in our patients. Second AAR is influenced by body mass index and sex as well. Both serum AST and ALT increase with body weight; however, this seems to be more prominent for ALT rather than AST. Furthermore, the AAR seems to be higher in women. Although both possible confounders were included in our regression analysis, AAR still remained significantly associated with CLI in our cohort.
Interestingly hypertension was associated with an OR of 0.7 with CLI in our regression analysis. We know that hypertension is an established risk factor for atherosclerosis; however, in this analysis it seems to be a protective factor for CLI. Possible explanations for this finding are the prescription of angiotensin-converting enzyme (ACE) or angiotensin II inhibitors, which might lead to a protective effect in PAOD patients. However, an increase in perfusion in the ischemic limb due to arterial hypertension might be protective for the development of CLI as well. In some centers, blood pressure levels in CLI patients are therefore allowed to be above recommended levels in order to increase perfusion of the ischemic limb until ulcer healing has occurred.
Our study has several drawbacks: first the retrospective study design. Second we used a single blood sample to calculate AAR. It therefore remains unclear whether this single blood sample reflects an elevated AAR over time. Third, one main reason for the close association of AAR with CLI might be the correlation of AAR with muscle-damage. AST is found (in a significantly higher concentration than ALT) in the muscle and can be released in case of muscular training or muscle damage, as is the case in CLI.[29–31] In this case, AST is more elevated than ALT and therefore the AAR is augmented. However, so far creatine kinase is still the enzyme of choice to evaluate muscular damage, as levels of creatine kinase in muscle are still higher than the levels of the AST and ALT.
However, we were able to show that AAR >1.67 can be used to discriminate patients at high risk for CLI from those with a low CLI-risk. Especially in combination with renal function, diabetes, and the age of the patients a discrimination of PAOD patients with high CLI risk seems possible.
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