Impact of Serum Zinc Level and Oral Zinc Supplementation on Clinical Outcomes in Patients Undergoing Infrainguinal Bypass for Chronic Limb-Threatening Ischemia

Abstract

Background: Zinc (Zn) has been reported to play an important role in wound healing (WH). Nevertheless, the effect of Zn in chronic limb-threatening ischemia (CLTI) patients is unclear. This study investigated the effect of Zn on the clinical outcomes of CLTI patients undergoing bypass surgery.

Methods and Results: This study reviewed 111 consecutive patients who underwent an infrainguinal bypass from 2012 to 2020. Patients with Zn deficiency (serum Zn level <60 μg/dL) received oral Zn supplementation and maintained a normal level until WH. This study aimed to explore: (1) the effect of Zn deficiency; and (2) Zn supplementation in Zn-deficient patients on the clinical outcomes of this cohort. Patients with Zn deficiency, Zn supplementation, and no Zn supplementation despite Zn deficiency accounted for 48, 21, and 42 patients, respectively. (1) Zn deficiency was associated with WH (HR, 0.47; 95% CI, 0.29–0.78: P=0.003), major adverse limb events (MALE) (HR, 2.53; 95% CI, 1.26–5.09: P=0.009), and major amputation or death (HR, 3.17; 95% CI, 1.51–6.63: P=0.002). (2) Zn supplementation was positively related to WH (HR, 2.30; 95% CI, 1.21–4.34: P=0.011). This result was confirmed using propensity score matching (HR, 2.24; 95% CI, 1.02–4.87: P=0.043).

Conclusions: The current study revealed that Zn level was associated with clinical outcomes in CLTI patients after bypass surgery. Oral Zn supplementation could improve WH in these patients.

Chronic limb-threatening ischemia (CLTI) is the most severe form of peripheral artery disease (PAD) and is characterized by ischemic resting pain and tissue loss. Revascularization is needed to relieve ischemic pain, heal ischemic wounds, prevent limb loss, and preserve ambulation and quality of life. Although the application of endovascular therapy (EVT) for CLTI has recently increased, surgical bypass using an autogenous vein plays an important role in the treatment of CLTI.¹ Several studies have recommended surgical bypass for appropriately selected patients because of its durable revascularization and superior wound healing rate compared to EVT.^2–4

Recently, several studies have reported that preoperative nutritional status is independently associated with the mortality risk after revascularization in patients with CLTI.^5,6 Several guidelines recommend that an interdisciplinary care team, including nutritionists/dietitians, evaluate and provide comprehensive care for patients with CLTI;^1,7 however, the efficacy of nutritional interventions in patients with complicated CLTI is not well understood.

Zinc (Zn), one of the essential micronutrients for humans, is involved in numerous biological functions and is considered a multipurpose trace element due to its capacity to bind to more than 300 enzymes and more than 2,000 transcription factors.⁸ It has also been reported to play an important role in wound healing.⁹ In addition, several reports have recently reinforced the association between Zn deficiency and cardiovascular disease, especially atherosclerosis.^10,11 Although CLTI seems to be one of the most severe atherosclerotic diseases, little has been reported on the relationship between CLTI and Zn deficiency. Furthermore, the effect of Zn deficiency on wound healing and other clinical outcomes in CLTI patients remains unclear. Therefore, the aim of the current study was to investigate the association of Zn deficiency with clinical outcomes in CLTI patients who underwent bypass surgery and, if there was an association, to examine whether oral supplementation with Zn could improve the clinical outcomes.

Methods

Subjects

This study was a retrospective analysis of prospectively collected data from consecutive patients who underwent de novo infrainguinal bypass surgery for ischemic tissue loss or gangrene with Wound, Ischemia, and foot Infection (WIfI) grade 2/3 ischemia due to arteriosclerosis obliterans and had Zn data from May 2012 to August 2020, at the Division of Vascular and Endovascular Surgery, Department of Surgery, Nagoya University Graduate School of Medicine, and Division of Vascular Surgery, Ichinomiya Municipal Hospital.¹²

The serum Zn level was measured in the morning on the day before bypass surgery and was monitored every 2–4 weeks until wound healing. The cut-off for Zn deficiency was defined as 60 μg/dL, according to Japanese clinical guidelines for Zn deficiency.¹³ From May 2012 to December 2017, the serum Zn level was measured at the discretion of each vascular surgeon. During this period, even though the Zn level was low, only 1 patient took a Zn supplement. From January 2018 to August 2020, the Zn level was measured in consecutive patients who planned to undergo bypass surgery. When the serum Zn level was <60 μg/dL, a patient received oral Zn supplementation (Zn acetate hydrate [Nobelzin®, Nobelpharma Co., Ltd. Tokyo, Japan] with 50 mg of elemental Zn every day) and maintained a normal level until wound healing.

In the present study, patients were divided into 3 groups: the Zn sufficiency group (serum Zn level ≥60 μg/dL: group S), the Zn supplementation for deficiency group (preoperative serum Zn level <60 μg/dL and supplementation after bypass: group SD), and the no Zn supplementation despite deficiency group (preoperative serum Zn level <60 μg/dL but no Zn supplementation: group NSD).

This study was conducted in accordance with the mandates of the Declaration of Helsinki. The Nagoya University School of Medicine Institutional Review Board approved the study (approval number: 2020-0074), and all patients provided written informed consent prior to surgery and data collection.

Outcome Measures

The primary endpoint was wound healing after infrainguinal bypass. The secondary endpoints included freedom from major adverse limb events (MALE) (i.e., major amputation or major reintervention), limb salvage (i.e., freedom from major amputation), amputation-free survival (i.e., freedom from the composite outcome of major amputation and all-cause mortality), and overall survival (i.e., freedom from all-cause mortality).¹

Definitions

Coronary artery disease (CAD) was defined as a history of any revascularization of the coronary arteries. Cerebrovascular disease (CVD) was defined as a history of stroke, cerebral hemorrhage, and/or any revascularization of the carotid arteries. Diabetes mellitus (DM) was diagnosed in patients who were taking hypoglycemic drugs or who self-injected insulin. Complete wound healing was defined as complete epithelialization of all wounds on the affected limbs without death or major amputation.¹⁴ The duration from the initial bypass surgery to complete epithelialization was defined as the wound healing time. The WIfI stages of patients before the original paper describing the WIfI staging system was published were retrospectively determined using photographs of pedal wounds and medical records, including laboratory findings on revascularization by an independent surgeon.¹² The Global Limb Anatomic Staging System (GLASS) stage before the global vascular guideline was published was also retrospectively determined using digital subtraction angiograms by vascular surgeons blinded to the clinical outcomes.¹

Revascularization Procedure and Follow up

All CLTI patients at both institutions were generally considered for revascularization. The authors performed angioplasty and surgical bypass. The treatment of all patients was discussed among vascular surgeons, and the best revascularization strategy was determined after consideration of each patient’s comorbidities and activities of daily living, the availability of a suitable venous conduit, the anatomical features of arterial disease, and the severity of ischemic wounds. In many cases, CLTI patients exhibit extensive atherosclerosis and long segments of occlusion; therefore, bypass surgery is preferred as a first-line treatment, especially for infrapopliteal lesions.¹⁵ We generally perform EVT in cases of Trans-Atlantic Inter-Society Consensus II (TASCII) A, B and C disease and perform bypass surgery in cases of TASCII D disease for the revascularization of femoropopliteal lesions.¹⁶ We did not perform bypass surgery in patients with severe dementia, those who were bedridden long term because of neurogenic deficits, those with extensive infection above the ankle, or those who refused revascularization.

Wound Care

Wound care was standardized according to the Tissue, Inflammation/infection, Moisture imbalance, Epithelial edge advancement (TIME) concept at both institutions.¹⁷ After wound irrigation, an assessment was performed. If the examination alone was insufficient or the wound defect was extensive (e.g., exposed bone, necrosis), sharp excisional debridement was routinely performed after revascularization. The wound was covered with dressing materials suitable for the wound condition. Systemic antibiotics were prescribed only in cases of infection at the wound site or for other lesions. Negative-pressure wound therapy was also used in cases with sufficient blood flow to the wound and no infection at the discretion of the physician. After patient discharge, dermatologists, plastic surgeons, or nurses cared for the wound every day or every other day. The authors also continued wound care every 2–3 weeks at an outpatient clinic.

Statistical Analysis

Unless otherwise stated, data are expressed as the mean±standard deviation for continuous variables and as percentages for dichotomous variables. Values without normal distribution were presented as median [interquartile range, IQR, 25th–75th percentile]. When comparing 2 groups, the unpaired t-test or the Wilcoxon rank-sum test was used for the univariate analysis of continuous data, depending on the distribution of the data. The chi-squared test was used for the univariate analysis of categorical data. Long-term clinical outcomes were analyzed using Kaplan-Meier survival curves and then compared statistically using the log-rank test. Univariate analysis was used to identify potential predictors of long-term clinical outcomes, and covariates with P<0.05 were entered into a multivariate Cox proportional hazards regression analysis. Clinical variables found to influence wound healing in previous studies were included in the univariate analysis.^18,19

First, group S was compared with group NSD to clarify the effect of Zn deficiency on clinical outcomes. Second, among patients with Zn deficiency, group NSD was compared with group SD. To clarify the effect of Zn supplementation on clinical outcomes, these comparisons were also analyzed after propensity score matching to minimize background differences between the 2 groups. The propensity score analysis was derived from a logistic regression model including sex and comorbidities (including hemodialysis-dependent, WIfI clinical stage). Matching was based on the logit of the propensity score, within a caliper of 0.25 SD of the value. Furthermore, as 1 : 1 propensity score matching typically eliminates a substantial number of cases from the analysis, inverse probability of treatment weighting (IPTW) was also applied for sensitivity analysis.²⁰ IPTW is a form of propensity score analysis that achieves balance in the distribution of covariates by weighting all observations on the inverse of the propensity score to create a well-balanced pseudo-cohort. Comparisons after matching and IPTW were performed using weighted Kaplan-Meier survival curves and the Cox model. A P value <0.05 was considered statistically significant. Statistical analyses were performed using the Statistical Package for Social Sciences software (SPSS version 27; SPSS, Inc., Chicago, IL, USA) and R (version 4.0.2; The R Foundation).

Results

Study Population and Patient Characteristics

From May 2012 to August 2020, 149 patients underwent de novo infrainguinal bypass for CLTI. We excluded patients with resting pain (n=13) and without data regarding the serum Zn level (n=25). Finally, consecutive patients (n=111) with data regarding the Zn level at baseline and regular intervals after bypass surgery were included in the current study. There were 48, 21, and 42 patients in group S, SD, and NSD, respectively (Supplementary Figure 1).

The overall baseline characteristics of the study population are summarized in Table 1. The mean age was 72 years, and the prevalence of DM and hemodialysis-dependent renal failure was 74% and 54%, respectively. WIfI clinical stage 1, 2, 3, and 4 accounted for 0%, 1%, 25%, and 74%, respectively. Distal anastomosis was performed on crural or pedal arteries in 66 patients (59%). As summarized in Table 1, the background characteristics were basically similar between group S and group NSD, except for CVD, serum albumin level, and the WIfI ischemia grade, and were similar between group NSD and group SD, except for CAD and the WIfI ischemia grade.

Table 1. Comparison of the Baseline Characteristics of the Study Population

	Zn sufficiency (group S: n=48)	Zn deficiency (n=63)		P value
		No Zn supplementation (group NSD: n=42)	Zn supplementation (group SD: n=21)	Group S vs. group NSD	Group NSD vs. group SD
Patient status
Male	29 (60)	29 (69)	14 (67)	0.51	1.00
Age (years)	72±7	72±9	73±8	0.93	0.88
HT	35 (73)	34 (81)	17 (81)	0.46	1.00
DL	28 (58)	19 (45)	5 (24)	0.29	0.17
DM	38 (79)	28 (67)	16 (76)	0.23	0.56
CAD	34 (71)	25 (60)	18 (86)	0.28	0.05
HD	23 (48)	23 (55)	14 (67)	0.53	0.42
CVD	14 (29)	4 (10)	2 (10)	0.03	1.00
Baseline albumin level (g/dL)	3.4±0.7	3.0±0.6	3.1±0.5	0.02	0.79
Baseline Zn level (μg/dL)	71±11	50±6	49±7	<0.001	0.35
Limb status
GLASS stage (I/II/III)	3 (6)/6 (13)/ 39 (81)	3 (7)/10 (24)/ 29 (69)	1 (5)/4 (19)/ 16 (76)	0.35	0.83
WIfI wound grade (1/2/3)	19 (40)/17 (35)/ 12 (25)	13 (31)/18 (43)/ 11 (26)	4 (19)/11 (52)/ 6 (29)	0.67	0.59
WIfI ischemia grade (2/3)	8 (17)/40 (83)	1 (2)/41 (98)	6 (29)/15 (71)	0.03	0.004
WIfI foot infection grade (0/1/2/3)	12 (25)/18 (38)/ 16 (33)/2 (4)	14 (33)/15 (36)/ 9 (21)/4 (10)	3 (14)/8 (38)/ 10 (48)/0 (0)	0.45	0.07
WIfI stage (2/3/4)	1 (2)/14 (29)/ 33 (69)	0 (0)/11 (26)/ 31 (74)	0 (0)/3 (14)/ 18 (86)	0.60	0.35
Outflow
Popliteal	18 (38)	17 (40)	10 (48)	0.30	0.64
Crural	20 (42)	21 (50)	8 (38)
Pedal	10 (20)	4 (10)	3 (14)

Data are presented as n (%) or the mean±standard deviation. CAD, coronary artery disease; CVD, cerebrovascular disease; DL, dyslipidemia; DM, diabetes mellitus; GLASS, Global Limb Anatomic Staging System; HD, hemodialysis; HT, hypertension; NSD, preoperative serum Zn level <60 μg/dL but no Zn supplementation; SD, preoperative serum Zn level <60 μg/dL and supplementation after bypass; WIfI, Wound, Ischemia, and foot Infection; Zn, zinc.

Clinical Outcomes in Patients With Normal and Low Zn Levels

The median duration of follow up was 707 days [IQR, 178–1,151] in this study. As shown in Figure 1A, the wound healing rate in group NSD was significantly lower than that in group S (62% in group NSD and 92% in group S at 1 year after bypass surgery; log-rank test, P<0.001). Freedom from MALE, limb salvage, amputation-free survival, and overall survival were also significantly worse in group NSD than in group S (Figure 1B–E).

Figure 1.

Comparison between the zinc (Zn) sufficiency group (serum Zn level ≥60 μg/dL: group S), and the no Zn supplementation despite deficiency group (preoperative serum Zn level <60 μg/dL but no Zn supplementation: group NSD) regarding long-term clinical outcomes in patients who underwent bypass surgery for chronic limb-threatening ischemia (CLTI): (A) wound healing rate, (B) freedom from major adverse limb event (MALE), (C) limb salvage, (D) amputation-free survival, and (E) overall survival.

The associations of baseline characteristics with wound healing and other clinical outcomes (MALE, major amputation or death, and overall mortality) are shown in Table 2. Hemodialysis-dependent renal failure (hazard ratio [HR], 0.34; 95% confidence interval [CI], 0.30–0.96: P=0.004), Zn deficiency (HR, 0.53; 95% CI, 0.31–0.89: P=0.002), and WIfI wound grade (HR, 0.63; 95% CI, 0.44–0.89: P=0.008) were negatively associated with wound healing. Zn deficiency was also independently related to MALE (HR, 2.53; 95% CI, 1.26–5.09: P=0.009), major amputation (HR, 6.43; 95% CI, 1.82–22.74: P=0.004), and major amputation or death (HR, 3.17; 95% CI, 1.51–6.63: P=0.002). Regarding major amputation, univariate regression analysis revealed that hemodialysis, Zn deficiency, the WIfI wound grade, and the WIfI foot infection grade were independent predictors. However, the number of cases of major amputation was quite limited (n=17), and multiple regression analysis using a Cox proportional hazards model might not have been reliable because of a lack of statistical power.

Table 2. Relationship Between Baseline Patient/Limb Characteristics and Clinical Outcomes Following Bypass Surgery in CLTI Patients

Outcome measures / Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Wound healing (n=67)
Age (>72 years)	0.93	0.40–2.17	0.87
Male (vs. female)	0.82	0.50–1.34	0.43
HT	1.01	0.57–1.80	0.96
DL	1.27	0.79–2.06	0.33
DM	0.97	0.57–1.65	0.90
CAD	0.88	0.53–1.44	0.60
HD	0.43	0.26–0.70	<0.001	0.35	0.30–0.96	0.004
CVD	0.88	0.49–1.58	0.66
Baseline albumin level	2.36	1.62–3.43	<0.001	1.41	0.90–2.20	0.13
Zn deficiency	0.43	0.26–0.72	0.001	0.53	0.31–0.89	0.002
GLASS stage	0.89	0.60–1.32	0.56
WIfI wound grade	0.56	0.41–0.77	<0.001	0.63	0.44–0.89	0.008
WIfI ischemia grade	0.62	0.31–1.26	0.19
WIfI foot infection grade	0.74	0.56–0.98	0.04	1.02	0.75–1.40	0.91
Major adverse limb event (n=35)
Age (>72 years)	0.74	0.38–1.44	0.37
Male (vs. female)	0.74	0.38–1.46	0.39
HT	1.47	0.61–3.55	0.39
DL	0.97	0.50–1.89	0.93
DM	0.74	0.36–1.51	0.40
CAD	1.47	0.70–3.09	0.30
HD	2.28	1.14–4.59	0.02	2.01	0.99–4.08	0.05
CVD	1.23	0.56–2.70	0.61
Zn deficiency	2.33	1.18–4.59	0.02	2.53	1.26–5.09	0.009
GLASS stage	1.55	0.78–3.07	0.21
WIfI wound grade	1.97	1.27–3.05	0.002	2.06	1.30–3.25	0.002
WIfI ischemia grade	1.30	0.40–4.25	0.66
WIfI foot infection grade	1.14	0.80–1.63	0.47
Major amputation (n=17)
Age (>72 years)	1.29	0.49–3.39	0.61
Male (vs. female)	1.26	0.45–3.59	0.66
HT	1.32	0.38–4.59	0.66
DL	0.60	0.23–1.56	0.29
DM	1.80	0.52–6.27	0.36
CAD	1.03	0.38–2.78	0.96
HD	5.79	1.65–20.34	0.01	3.23	0.87–11.98	0.08
CVD	0.52	0.12–2.28	0.39
Zn deficiency	6.42	1.84–22.37	0.004	6.43	1.82–22.74	0.004
GLASS stage	1.78	0.61–5.20	0.30
WIfI wound grade	2.61	1.34–5.08	0.01	2.09	0.96–4.55	0.06
WIfI ischemia grade	1.99	0.26–15.00	0.51
WIfI foot infection grade	2.02	1.19–3.44	0.01	1.60	0.88–2.99	0.13
Major amputation or death (n=36)
Age (>72 years)	0.85	0.44–1.67	0.64
Male (vs. female)	1.13	0.55–2.31	0.74
HT	1.13	0.49–2.59	0.78
DL	0.53	0.27–1.04	0.06
DM	1.55	0.70–3.45	0.28
CAD	0.99	0.50–1.97	0.98
HD	3.86	1.82–8.18	<0.001	3.40	1.55–7.45	0.002
CVD	0.89	0.39–2.03	0.77
Zn deficiency	3.06	1.50–6.23	0.002	3.17	1.51–6.63	0.002
GLASS stage	1.15	0.63–2.10	0.64
WIfI wound grade	1.77	1.15–2.73	0.01	1.90	1.19–3.03	0.008
WIfI ischemia grade	3.94	0.54–28.81	0.18
WIfI foot infection grade	1.41	0.99–2.04	0.06
Overall mortality (n=31)
Age (>72 years)	0.60	0.29–1.26	0.18
Male (vs. female)	1.46	0.65–3.28	0.36
HT	1.27	0.51–3.12	0.61
DL	0.51	0.25–1.06	0.07
DM	2.33	0.89–6.12	0.09
CAD	1.13	0.53–2.38	0.76
HD	3.91	1.76–8.73	<0.001	3.15	1.37–7.24	0.007
CVD	0.89	0.36–2.19	0.80
Zn deficiency	2.40	1.13–5.09	0.02	1.90	0.88–4.12	0.10
GLASS stage	1.27	0.65–2.46	0.49
WIfI wound grade	1.62	1.03–2.57	0.04	1.42	0.88–2.29	0.15
WIfI ischemia grade	23.84	0.11–5,190.21	0.25
WIfI foot infection grade	1.29	0.89–1.87	0.17

CI, confidence interval; CLTI, chronic limb-threatening ischemia; HR, hazard ratio; n, number of observed events. Other abbreviations as in Table 1.

Along with Zn deficiency, the WIfI wound grade was also associated with MALE (HR, 2.06; 95% CI, 1.30–3.25: P=0.002) and major amputation or death (HR, 1.90; 95% CI, 1.19–3.03: P=0.008). Hemodialysis was associated with major amputation or death (HR, 3.40; 95% CI, 1.55–7.45: P=0.002) and overall mortality (HR, 3.15; 95% CI, 1.37–7.24: P=0.007).

Clinical Outcomes After Zn Supplementation in Patients With Zn Deficiency

Data of patients with Zn supplementation for deficiency (group SD, n=21) and without Zn supplementation despite Zn deficiency (group NSD; n=42) were analyzed. In group SD, the serum Zn level increased at 1 month after surgery in 20/21 (95%) of patients. The median increases in Zn levels at 1 month, and 3 months were 23 μg/dL [IQR, 11–43], and 32 μg/dL [IQR, 15–40], respectively. The patient who did not have an increase in Zn levels despite Zn administration was an 85-year-old man with DM, CAD, dyslipidemia, and WIfI stage 4 (Wound/Ischemia/foot Infection grade=2/3/4). He achieved wound healing on 34 postoperative day (POD). Although there should be several explanations for this outcome, ischemic wounds with low WIfI wound grades might be one of the reasons.

During the study period, no side-effects of Zn supplementation were observed. The cumulative wound healing rate was significantly better in group SD than in group NSD (P=0.041, Figure 2A). Similarly, the cumulative rates of freedom from MALE (P=0.011, Figure 2B) and limb salvage (P=0.021, Figure 2C) were also significantly better in group SD than in group NSD. In contrast, the rates of amputation-free survival (P=0.20, Figure 2D) and overall survival (P=0.68, Figure 2E) were not significantly different between the groups.

Figure 2.

Comparison between the zinc (Zn) supplementation for deficiency group (preoperative serum Zn level <60 μg/dL and supplementation after bypass: group SD), and the no Zn supplementation despite deficiency group (preoperative serum Zn level <60 μg/dL but no Zn supplementation: group NSD) regarding long-term clinical outcomes in patients who underwent bypass surgery for chronic limb-threatening ischemia (CLTI): (A) wound healing, (B) freedom from major adverse limb event (MALE), (C) limb salvage, (D) amputation-free survival, and (E) overall survival in patients with perioperative Zn deficiency who underwent bypass surgery.

The associations of baseline characteristics with wound healing and other clinical outcomes (MALE, major amputation or death, and overall mortality) are shown in Table 3. Zn supplementation was positively related to wound healing (HR, 2.21; 95% CI, 1.16–4.21: P=0.02) and negatively associated with MALEs (HR, 0.17; 95% CI, 0.05–0.56: P=0.004). In contrast, the WIfI wound grade was associated with MALE (HR, 2.28; 95% CI, 1.25–4.16: P=0.007), major amputation (HR, 2.40; 95% CI, 1.03–5.58: P=0.04), and major amputation or death (HR, 2.75; 95% CI, 1.37–5.50: P=0.004).

Table 3. Relationship Between Baseline Patient/Limb Characteristics and Clinical Outcomes Following Bypass Surgery in CLTI Patients With Zn Deficiency

Outcome measures / Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
Wound healing (n=45)
Age (>72 years)	0.98	0.55–1.76	0.94
Male (vs. female)	1.07	0.57–2.01	0.84
HT	0.73	0.35–1.51	0.39
DL	0.94	0.52–1.73	0.85
DM	0.81	0.43–1.53	0.51
CAD	0.96	0.51–1.80	0.90
HD	0.46	0.25–0.82	0.01	0.62	0.0–1.26	0.12
CVD	0.43	0.13–1.38	0.16
Baseline albumin level	2.14	1.34–3.42	0.002	1.31	0.74–2.34	0.36
Zn supplementation	1.85	1.01–3.36	0.05	2.21	1.16–4.21	0.02
GLASS stage	0.97	0.62–1.51	0.88
WIfI wound grade	0.54	0.37–0.81	0.002	0.71	0.44–1.12	0.14
WIfI ischemia grade	1.04	0.41–2.63	0.94
WIfI foot infection grade	0.68	0.49–0.95	0.02	0.78	0.55–1.11	0.18
Major adverse limb event (n=24)
Age (>72 years)	0.74	0.33–1.66	0.47
Male (vs. female)	1.14	0.47–2.75	0.78
HT	1.17	0.40–3.44	0.78
DL	1.55	0.69–3.45	0.29
DM	0.81	0.34–1.97	0.64
CAD	0.83	0.36–1.91	0.66
HD	2.61	1.03–6.62	0.04	2.45	0.94–6.36	0.07
CVD	1.16	0.35–3.91	0.81
Zn supplementation	0.24	0.07–0.80	0.02	0.17	0.05–0.56	0.004
GLASS stage	1.33	0.64–2.75	0.44
WIfI wound grade	2.13	1.18–3.85	0.01	2.28	1.25–4.16	0.01
WIfI ischemia grade	1.53	0.36–6.52	0.57
WIfI foot infection grade	1.44	0.91–2.30	0.12
Major amputation (n=15)
Age (>72 years)	1.03	0.37–2.83	0.96
Male (vs. female)	1.27	0.40–3.99	0.68
HT	0.61	0.19–1.91	0.40
DL	1.67	0.61–4.63	0.32
DM	1.67	0.47–5.91	0.43
CAD	0.97	0.33–2.83	0.95
HD	3.32	0.93–11.86	0.06
CVD	1.45	0.33–6.45	0.62
Zn supplementation	0.13	0.02–1.00	0.05
GLASS stage	1.97	0.64–6.07	0.24
WIfI wound grade	3.02	1.35–6.74	0.01	2.40	1.03–5.58	0.04
WIfI ischemia grade	24.59	0.03–22,554.74	0.36
WIfI foot infection grade	2.14	1.16–3.95	0.02	1.74	0.89–3.39	0.11
Major amputation or death (n=28)
Age (>72 years)	0.93	0.44–1.96	0.85
Male (vs. female)	1.22	0.54–2.76	0.63
HT	1.22	0.42–3.51	1.22
DL	0.69	0.32–1.52	0.36
DM	1.41	0.59–3.35	0.44
CAD	1.12	0.51–2.45	0.79
HD	3.01	1.27–7.12	0.01	2.35	0.96–5.75	0.06
CVD	0.83	0.19–3.55	0.80
Zn supplementation	0.53	0.20–1.42	0.21
GLASS stage	1.91	0.87–4.22	0.11
WIfI wound grade	3.15	1.68–5.91	<0.001	2.75	1.37–5.50	0.004
WIfI ischemia grade	0.93	0.28–3.13	0.91
WIfI foot infection grade	1.76	1.13–2.73	0.01	1.22	0.74–2.02	0.44
Overall mortality (n=23)
Age (>72 years)	0.52	0.22–1.23	0.14
Male (vs. female)	1.50	0.60–3.75	0.38
HT	3.45	0.77–15.34	0.10
DL	0.44	0.18–1.08	0.07
DM	2.04	0.69–6.02	0.20
CAD	1.51	0.63–3.61	0.36
HD	2.88	1.12–7.44	0.03	2.04	0.73–5.73	0.18
CVD	0.39	0.05–2.91	0.36
Zn supplementation	0.79	0.25–2.45	0.68
GLASS stage	2.63	0.99–6.91	0.05
WIfI wound grade	2.02	1.13–3.61	0.02	1.65	0.87–3.15	0.13
WIfI ischemia grade	0.52	0.15–1.83	0.31
WIfI foot infection grade	1.47	0.96–2.27	0.08

Abbreviations as in Tables 1,2.

After adjustment using propensity score matching, the two groups were determined to be well balanced in terms of all variables (Supplementary Table). As shown in Figure 3A,B, the rates of wound healing and freedom from MALE were significantly better in group SD than in group NSD (P<0.001 and P<0.001, respectively). Zn supplementation was associated with better wound healing (HR, 3.93; 95% CI, 1.68–9.23: P=0.002) and freedom from MALE (HR, 0.15; 95% CI, 0.04–0.53: P=0.003). The IPTW-adjusted rates of wound healing and freedom from MALE were also significantly better in group SD than in group NSD (Supplementary Figure 2A,B). Moreover, Zn supplementation was also associated with better wound healing (HR, 2.18; 95% CI, 1.27–3.73: P=0.005) and freedom from MALE (HR, 0.15; 95% CI, 0.04–0.58: P=0.006) with the use of IPTW.

Figure 3.

Comparison between the zinc (Zn) supplementation for deficiency group (preoperative serum Zn level <60 μg/dL and supplementation after bypass: group SD), and the no Zn supplementation despite deficiency group (preoperative serum Zn level <60 μg/dL but no Zn supplementation: group NSD) after propensity score matching analysis regarding long-term clinical outcomes in patients who underwent bypass surgery for chronic limb-threatening ischemia (CLTI): (A) wound healing and (B) freedom from major adverse limb event (MALE) in patients with perioperative Zn deficiency who underwent bypass surgery after propensity score analysis.

Discussion

The current study investigated whether the serum Zn level in CLTI patients affected their clinical outcomes after bypass surgery. To the best of our knowledge, this is the first study to show that: (1) a low serum Zn level is an independent predictor of wound healing and MALE; and (2) Zn supplementation in patients with a low serum Zn level is associated with wound healing and freedom from MALE.

Zn Deficiency and Wound Healing

Several possible explanations have been reported regarding the relationship between Zn deficiency and wound healing.^9,11 Generally, wound healing consists of 4 major stages: hemostasis, inflammation, proliferation, and remodeling.⁹ In the hemostasis phase, Zn can enhance platelet activity and aggregation, and initiate the inflammatory phase of wound healing. Moreover, Zn is an important coenzyme in the immune system and functions with cell proliferation-related enzymes involved in protein and DNA synthesis. In the remodeling phase, matrix metalloproteinase (MMP) family proteins, which are Zn-dependent endopeptidases, are essential for epidermal wound repair. Furthermore, Zn has been reported to be an antioxidant micronutrient.⁹ We recently reported that Zn deficiency impaired the rate of ischemia-induced revascularization through enhanced oxidative stress rates in mice experiments.²¹ It is well accepted that oxidative damage is a major cause of tissue injury and that redox regulation plays a prominent role in wound repair. Thus, Zn plays a significant role in regulating every phase of the wound healing process, affecting aspects ranging from membrane repair and oxidative stress to coagulation, inflammation, immunity, and remodeling.⁹

Although Zn is an essential trace element (micronutrient) that plays important roles in human physiology, and Zn deficiency has been reported to compromise wound healing, the relationship between Zn deficiency and wound healing in CLTI patients remains unclear.^8,9 Recently, we showed the relationship between Zn deficiency and clinical outcomes in CLTI patients undergoing infrainguinal bypass.²² Although this study demonstrated preliminary results regarding the effect of Zn deficiency on some clinical outcomes, it should be noted that the sample size was so small (n=43) that the multivariable models might be subject to overfitting. Moreover, although the Kaplan-Meier curve in this study showed a lower wound healing rate in the Zn deficiency group, there were significant differences in the patients’ background characteristics between the 2 groups (hemodialysis was more common in the Zn deficiency group), and these differences made it complicated to interpret this result. Furthermore, this preliminary study did not identify the predictors for wound healing. In the current study, as in the preliminary study, the baseline albumin level in group NSD was significantly lower than in group S.²² However, the baseline albumin level was not associated with wound healing after a multivariate analysis. Although this previous study showed novel findings, the results were based on a much smaller sample size than that of the present study.

Zn Deficiency and Arteriosclerotic Disease

Current clinical guidelines recommend screening for nutritional status during hospitalization and providing nutritional support for patients at risk of malnutrition.^23,24 Patients with preoperative Zn deficiency accounted for 43% (48/111) of the present cohort. Several reports have shown the prevalence of Zn deficiency in patients with cardiovascular disease.²⁵ Basically, a common cause of Zn deficiency is malnutrition, and Zn deficiency has been reported in the elderly, in patients with diabetes, and in patients on dialysis.^26–28 Several studies have determined that Zn deficiency in elderly people is due to a decline in dietary Zn intake with advancing age.²⁶ In diabetic patients, Zn excretion through urine is significantly increased by osmotic diuresis.²⁹ Zn deficiency in patients with chronic kidney disease is mainly due to a decrease in intestinal absorption. Furthermore, Zn is lost via the dialysis membrane, especially in patients on hemodialysis.³⁰ In general, a high proportion of CLTI patients are elderly and have these comorbidities.^3,31 In fact, in our cohort, the mean age was 72 years, and the prevalence of diabetes and dialysis was 74% and 54%, respectively. Therefore, clinicians who manage CLTI patients should take their serum Zn level into consideration. Patients with CLTI should undergo a nutritional assessment, including an evaluation for Zn deficiency.

Zn Supplementation vs. No Zn Supplementation in Zn-Deficient Patients

To date, no studies have investigated whether oral Zn supplementation accelerates wound healing or improves clinical outcomes in CLTI patients.³² In contrast, several studies have revealed the effect of Zn supplementation on wound healing in other populations, including patients with pressure ulcers and diabetic foot ulcers.^33,34 Although these studies provided notable findings, the effect of routine Zn supplementation on wound healing remains unclear.³⁵ Moreover, it has not been clarified whether these findings can be applied to patients with CLTI. In general, patients with CLTI are heterogeneous.¹ Even when assessing the severity of the limb threat, clinicians should use the WIfI classification system, which consists of the extent of the wound, degree of ischemia, and presence and severity of foot infection.¹ As such, the characteristics of patients and affected limbs vary widely. The current study performed analyses using both propensity score matching and IPTW to minimize the heterogeneity in the groups and obtain comparative treatment cohorts. Using this approach, we obtained well-balanced treatment groups, allowing comparison of long-term outcomes between treatment arms and eventually confirming the beneficial effects associated with Zn supplementation in Zn-deficient patients despite the low number of patients. However, the cause-and-effect relationship in the present study remains unknown. An intervention trial is needed to clarify whether Zn supplementation could promote wound healing and reduce the risk of MALE.

Study Limitations

First, this study was a retrospective, 2-center, observational study; therefore, there was selection bias for bypass surgery. We attempted to minimize the risk of bias by including all consecutive patients who met the inclusion criteria during the study period. Moreover, we compensated for the high probability of bias in many of the data assignments by using propensity-matched scores, as well as IPTW analysis for sensitivity analysis. We acknowledge, however, the possibility of residual confounding from both measured and unmeasured variables. Second, this study analyzed a low number of sample cases. Therefore, the results in the current study are likely vulnerable to type I or type II statistical errors, subject to overfitting in the multivariable models because of small samples and should be interpreted with caution. Third, the WIfI stage of each patient could be a potential bias, although data were prospectively collected and reviewed by an independent vascular surgeon blinded to the clinical results. Fourth, the selected procedure (ointment, maintenance debridement, etc.) for wound care and medication depended on each surgeon, although these decisions were all based on the TIME concept. Thus, these choices might have affected the clinical outcomes. Fifth, the effect of Zn supplementation on wound healing in CLTI patients without Zn deficiency is less understood. It remains unknown which patients with normal preoperative Zn levels will have a postoperative decrease in Zn levels, and whether these patients will have an improvement in clinical outcomes with Zn supplementation. Finally, the detailed mechanism underlying the observed differences is unclear. Although this study has several limitations, we believe that it demonstrates the importance of assessing the serum Zn level and providing Zn supplementation for Zn deficiency in the management of CLTI patients.

Conclusions

In the current study, we investigated: (1) the effect of Zn deficiency on clinical outcomes in CLTI patients who underwent bypass surgery; and (2) whether Zn supplementation was associated with improved clinical outcomes. Zn deficiency was negatively associated with wound healing and positively associated with MALE, major amputation, and major amputation or death. Furthermore, Zn supplementation was positively related to wound healing even after propensity score analysis. Zn could play a potential role in some clinical outcomes in CLTI patients undergoing bypass surgery. Prospective multicenter randomized control studies are warranted to confirm the findings of the present study.

Disclosures

K.K. is a member of Circulation Journal’s Editorial Team.

IRB Information

The Nagoya University School of Medicine Institutional Review Board approved the study (approval number: 2020-0074).

Data Availability

The deidentified participant data will not be shared.

Supplementary Files

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-21-0832

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