DOI: 10.1111/eci.12208

ORIGINAL ARTICLE Homoarginine and mortality in an older population: the Hoorn study Stefan Pilz*,†, Tom Teerlink‡, Peter G. Scheffer‡, Andreas Meinitzer§, Femke Rutters*, Andreas Tomaschitz¶, €rz§,§§,¶¶ and Christiane Drechsler**, Katharina Kienreich†, Giel Nijpels††, Coen D. A. Stehouwer‡‡, Winfried Ma Jacqueline M. Dekker* *Department of Epidemiology and Biostatistics, EMGO Institute for Health and Care Research, VU University Medical Centre, Amsterdam, the Netherlands, †Division of Endocrinology and Metabolism, Department of Internal Medicine, Medical University of Graz, Graz, Austria, ‡Metabolic Laboratory, Department of Clinical Chemistry and Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, the Netherlands, §Clinical Institute of Medical and Chemical Laboratory Diagnostics , ¶Division of Cardiology, Department of Internal Medicine, Medical University of Graz, Graz, Austria, € rzburg, Wu € rzburg, Germany, ††Department of General **Division of Nephrology, Department of Medicine, University of Wu Practice and the EMGO Institute for Health and Care Research, VU University Medical Centre, Amsterdam, the Netherlands, ‡‡ Department of Internal Medicine and Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, Maastricht, the Netherlands, §§Department of Medicine V (Nephrology, Hypertensiology, Endocrinlogy, Diabetology, Rheumatology), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany, ¶¶Synlab Academy, Synlab Services GmbH, Mannheim, Germany

ABSTRACT Background Homoarginine is an amino acid that may be involved in nitric oxide and energy metabolism. Previous studies in patient populations showed that low homoarginine levels indicate an increased risk of mortality and cardiovascular disease. We evaluated whether low plasma levels of homoarginine are associated with elevated, overall and cause-specific mortality. Materials and methods The Hoorn study is a population-based study among older men and women. We calculated Cox proportional hazard ratios (HRs) for overall and cause-specific mortality according to sex-specific homoarginine quartiles. Results We included 606 study participants (51
3% women; 70
0  6
6 years). Homoarginine concentrations were higher in men (1
63  0
51 lM), compared with women (1
30  0
44 lM; P < 0
001). After a median follow-up time of 7
8 years, 112 study participants died, including 31 deaths due to cardiovascular diseases and 30 due to cancer. Associations between homoarginine levels and mortality showed a threshold effect with a significant risk increase from the second to the first quartile. Compared with the upper three quartiles, the age-, sex- and BMI-adjusted HR (with 95% CI) in the first quartile was 2
26 (1
52–3
32) for overall mortality, 4
20 (2
03–8
69) for cardiovascular mortality and 1
25 (0
55–2
85) for cancer mortality. These associations remained materially unchanged after multivariate adjustments. Conclusions Low plasma concentrations of homoarginine are a risk marker for overall mortality and especially for cardiovascular mortality in the older general population. Further studies are warranted to elucidate the underlying pathophysiological mechanisms. Keywords AMINO acids, cardiovascular, homoarginine, mortality, prospective. Eur J Clin Invest 2014; 44 (2): 200–208

Introduction Accumulating evidence argues for a pivotal role of amino acids in metabolic and cardiovascular pathophysiology [1]. In this context, low levels of the amino acid homoarginine have been associated with a significantly increased risk of heart failure, stroke and mortality in various patient populations [2–7]. In

200

line with this, serum homoarginine was also significantly correlated with endothelial function in pregnant women [8]. The underlying mechanisms for the associations between homoarginine and cardiovascular risk remain to be elucidated, but experimental data suggest that homoarginine may modulate

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HOMOARGININE AND MORTALITY IN AN OLDER POPULATION

the metabolism of the vasodilator nitric oxide (NO) [9–13]. The significance and the direction of homoarginine effects on NO availability are however still unclear. In this context, some experimental data show that homoarginine inhibits platelet aggregation, increases insulin secretion and reduces blood pressure in rats [9–13]. Moreover, homoarginine is a well known inhibitor of alkaline phosphatase, which is in line with data on inverse associations between homoarginine and bone turnover [6,14]. It is also important to note that except of grass pea, lentil and similar legumes, a usual Western diet does not contain significant amounts of homoarginine [9,15]. Hence, endogenous synthesis from its precursor lysine seems to be the main source of homoarginine [15–17]. Interestingly, the proposed key enzyme for homoarginine synthesis, arginine:glycine amidinotransferase (AGAT), was found to be significantly upregulated in the myocardium of heart failure patients [18,19]. Considering that AGAT is crucial for the synthesis of the energy metabolite guanidinoacetate (GAA), which is further converted to creatine, it was hypothesised that increased AGAT expression in the heart aims to compensate for energy depletion in states of myocardial dysfunction [4,18,19]. Hence, there exists a reasonable rationale to hypothesise that homoarginine deficiency might be associated with cardiovascular risk and mortality. Previous observations addressing this issue were however exclusively carried out in patient cohorts, warranting further confirmation in population-based studies. We therefore measured homoarginine levels in the Hoorn study, a prospective cohort study among older men and women in the Netherlands. Interestingly, previous crosssectional analyses of this cohort showed that homoarginine was positively associated with blood pressure [20]. This finding might suggest some potentially adverse effects of high homoarginine levels, pointing to the urgent need to further elucidate the relationship between homoarginine and clinical outcomes in this population-based study. We therefore aimed to evaluate whether homoarginine levels are associated with overall and cause-specific mortality.

Materials and methods Study population The Hoorn study is a prospective population-based study among the older general population in the city of Hoorn in the Netherlands. The study started in 1989 and included 2484 men and women at the age of 50–75 years. The current work includes study subjects that participated in a follow-up examination in the year 2000–2001 and who were then prospectively followed up with respect to mortality. Detailed descriptions of all study procedures of this study visit, which is considered as the baseline for the present investigation, have been previously published [20–23]. In brief, all patients with type 2 diabetes

(n = 176) and a random sample of individuals with either normal (n = 705) or impaired glucose metabolism were invited for this examination in 2000–2001, of whom 648 (60%) participated. After the exclusion of individuals with missing homoarginine values (n = 42), we finally included the remaining 606 study participants in our present analysis. All participants gave written informed consent prior to study inclusion, and the study protocol was approved by the Ethics Committee of the VU University Medical Centre, Amsterdam.

Laboratory methods We determined the concentrations of homoarginine, arginine, symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA) in plasma by the use of high-performance liquid chromatography with fluorescence detection as previously described, with slightly modified chromatographic conditions [24,25]. For all these variables, the intraassay and inter-assay coefficient of variation (CV) was below 2
0% and 4
0%, respectively. Plasma concentrations of B-type natriuretic peptide (BNP) were measured by an immunoradiometric assay kit (Shionoria, Osaka, Japan) with intra- and inter-assay CV < 10%. Further laboratory measurements were performed according to routine methods as described elsewhere [20–23]. Glomerular filtration rate (GFR) was calculated according to the 4-variable equation from the modification of diet in renal disease (MDRD) study [26]. Diabetes mellitus was classified according to the 2010 criteria of the American Diabetes Association (ADA) as fasting glucose ≥ 7
0 mM, 2 h postload glucose ≥ 11
1 mM or HbA1c ≥ 6
5%. Participants on glucose-lowering drugs were also classified as patients with diabetes mellitus. Arterial hypertension was diagnosed in individuals with systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg or who use antihypertensive drugs.

Follow-up procedures Survival data were obtained from the municipal register of the city of Hoorn [23]. Causes of death were determined according to medical records from general practitioners or local hospitals. The ninth edition of the International Classification of Diseases (ICD-9) was used for the coding of death causes. Based on this, cardiovascular mortality was classified in deceased patients with ICD codes 390–459 (diseases of the circulatory system) or with ICD code 798 (sudden death). Cancer mortality was classified for ICD codes 140–239. Follow-up time was defined as the time between the baseline examination until the date of death or until the censoring date (1 January 2009). No study participant was lost during follow-up.

Statistical analysis Considering that previous studies reported significantly higher homoarginine concentrations in men compared with women,

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we formed sex-specific homoarginine quartiles [2]. Depending on their distribution, continuous data are either presented as means  standard deviation (SD) (normally distributed variables) or as medians with interquartile ranges (skewed variables). Variables following a non-normal distribution were log (e) transformed before use in parametric statistical analyses. Groups were compared using chi-square tests with P for linear by linear trend for categorical variables and by ANOVA with P for linear trend for continuous variables. Linear and binary logistic regression analyses adjusted for age and sex were used to evaluate whether homoarginine is associated with clinical and laboratory baseline variables. Cox proportional hazard ratios (HRs) for overall and cause-specific mortality were calculated according to sex-specific homoarginine quartiles using the fourth quartile as the reference. We calculated crude HRs, ageand sex-adjusted, as well as age-, sex- and body mass index (BMI)-adjusted HRs. In addition, we adjusted for various possible confounders as indicated. For the mortality analyses, we also tested for possible interactions of homoarginine quartiles with gender, diabetes and arterial hypertension by adding product terms to our Cox proportional hazard models. Assumptions for the Cox proportional hazards models were evaluated by logminus-log survival and partial (Sch€ onfeld) residuals vs. survival time plots and found valid. In addition, Kaplan–Meier curves followed by log-rank tests were calculated to graphically display the association of sex-specific homoarginine quartiles with mortality. The SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses. A P-value below 0
05 was considered to indicate statistical significance.

Results Our study cohort consisted of 606 participants (51
3% females), with a mean age of 70
0  6
6 years and mean homoarginine concentrations of 1
46  0
50 lM. There was a significant gender difference in homoarginine levels, with significantly higher concentrations in men (1
63  0
51 lM) compared with women (1
30  0
44 lM; P < 0
001). In addition, we observed an inverse correlation between homoarginine and age (Pearson correlation coefficient = 0
11; P = 0
009). Baseline characteristics according to sex-specific homoarginine quartiles are presented in Table 1. For all continuous variables in Table 1, we performed linear regression analyses using homoarginine, age and sex as the independent variables. In these analyses, significant results, presented as standardised beta coefficients for homoarginine, were obtained for associations of homoarginine with BMI (0
22; P < 0
001), waist to hip ratio (0
07; P = 0
033), systolic blood pressure (0
16; P < 0
001), diastolic blood pressure (0
13; P = 0
003), fasting glucose (0
14; P < 0
001), 2 h glucose (0
17; P < 0
001), HbA1c (0
12; P = 0
007), BNP ( 0
11; 0
013), serum

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albumin (0
14; P = 0
002), SDMA ( 0
11; P = 0
005) and arginine (0
34; P < 0
001). Similarly, we performed binary logistic regression analyses adjusted for age and sex, to test for associations of homoarginine levels with all categorical variables of Table 1. In these analyses, significant associations, presented as odds ratios (OR) with 95% CI for homoarginine (in lM), were found with arterial hypertension (OR: 1
81; 1
21–2
71; P = 0
004), ex- and active smokers (OR: 0
65; 0
45–0
94; P = 0
021) and lipidlowering drugs (OR: 1
59; 1
03–2
44; P = 0
036). Apart from the above-reported associations of homoarginine with age and gender, there were no further significant results in these linear and binary logistic regression analyses. After a median follow-up time of 7
8 years (interquartile range: 7
6–8
2 years), 112 study participants died. Sufficient information for the classification of specific causes of death was available in 87 (78%) of the deceased individuals. Of these, 31 (36%) died due to cardiovascular diseases and 30 (34%) due to cancer. Compared to the 4th sex-specific homoarginine quartile, the age-, sex-, and BMI-adjusted Cox proportional HRs (with 95% confidence intervals) for all-cause mortality in the 1st, 2nd and 3rd quartile were 1
87 (1
14–3
05), 0
70 (0
39–1
28) and 0
79 (0
44–1
41), respectively (Table 2). These associations were not substantially altered after adjustments for various covariates (see Table 2). In general, the relationship between homoarginine and mortality was slightly J-shaped with an indication for a strong threshold effect from the 2nd to the 1st quartile and no significant difference for mortality risk between the upper three quartiles (see Table 2). Considering this and taking into account the relatively low number of cause-specific deaths, we performed additional analyses for all-cause, cardiovascular and cancer mortality by calculating HRs for the first compared with the upper three quartiles. Depending on the statistical adjustments, the HRs for all-cause mortality ranged from 2
0 to 2
6 in the first sex-specific homoarginine quartile (see Table 3). An even more pronounced result was obtained for cardiovascular deaths, with an approximately four- to fivefold higher cardiovascular mortality in the first compared with the upper three quartiles (Table 3). Cancer mortality was however not significantly related to homoarginine levels (Table 3). There was no significant interaction with gender, diabetes mellitus and arterial hypertension in any of our analyses, which suggests that the association of homoarginine with outcome is not substantially altered by these variables. Kaplan–Meier curves for the first and the upper three sex-specific homoarginine quartiles are shown in Fig. 1 for all-cause mortality and in Fig. 2 for cardiovascular mortality. Log-rank tests showed that all-cause mortality and cardiovascular mortality were significantly increased in the first sex-specific homoarginine quartile (P < 0
001 for both). Additional analyses revealed that there was no significant association of arginine quartiles with any mortality outcome (data not shown).

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HOMOARGININE AND MORTALITY IN AN OLDER POPULATION

Table 1 Baseline characteristics according to sex-specific homoarginine quartiles Homoarginine (lM)

1st quartile 0
46–1
27

2nd quartile 1
01–1
55

3rd quartile 1
26–1
90

4th quartile 1
55–4
34

Number

152

153

151

150

P-value

Females (%)

51
3

51
0

51
7

51
3

0
968

Age (years)

71
3  6
9

69
9  6
5

69
3  6
7

69
5  6
2

0
013

Body mass index (kg/m²)

26
3  4
4

27
1  3
9

27
6  3
8

28
3  3
8

< 0
001

Waist to hip ratio

0
92  0
10

0
93  0
09

0
93  0
09

0
93  0
08

0
312

LDL cholesterol (mM)

3
5  0
9

3
8  0
9

3
7  0
9

3
5  0
9

0
638

HDL cholesterol (mM)

1
43  0
45

1
43  0
41

1
39  0
37

1
40  0
41

0
391

Triglycerides (mM)

1
3 (1
0–1
7)

1
4 (0
9–1
9)

1
3 (1
0–1
7)

1
3 (1
0–1
8)

0
606

Systolic BP (mmHg)

139  20

142  19

144  22

146  21

0
002

Diastolic BP (mmHg)

80  11

83  9

83  12

84  12

0
008

Arterial hypertension (%)

62
3

66
4

76
7

73
8

0
007

Fasting glucose (mM)

5
9 (5
4–6
4)

5
8 (5
4–6
5)

5
9 (5
5–6
5)

2 h glucose (mM)

6
8 (5
4–8
5)

6
4 (5
5–8
3)

6
8 (5
4–8
4)

7
6 (5
7–9
9)

0
013

HbA1c (%)

5
9 (5
6–6
3)

5
8 (5
5–6
2)

5
9 (5
6–6
2)

5
9 (5
7–6
4)

0
206

Diabetes mellitus (%)

6
0 (5
5–6
9)

0
029

28
9

30
1

23
2

32
0

0
895

GFR (mL/min/1
73 m )

59
2  10
4

59
1  9
7

60
8  10
2

59
3  9
8

0
631

Microalbuminuria (%)

15
8

20
4

11
9

12
1

0
130

2

C-reactive protein (mg/L)

2
41 (1
13–5
10)

1
85 (0
96–4
43)

1
90 (1
16–4
68)

2
32 (1
10–4
24)

0
493

BNP (pM)*

6
4 (3
8–14
4)

4
1 (1
9–7
3)

4
2 (1
8–9
1)

4
6 (2
0–8
8)

0
003

Heart rate (beats per min)

63
4  10
6

61
0  8
3

62
5  9
0

62
9  9
1

0
952

25-hydroxyvitamin D (nM)

52  21

57  22

54  18

52  17

0
802

PTH (pM) Serum albumin Homoarginine (lM) Arginine (lM)

5
7 (4
5–7
5) 41 (39–43) 0
93  0
20 87
9  13
1

5
5 (4
6–6
9) 41 (40–43) 1
27  0
17 93
6  13
6

5
6 (4
8–6
8) 42 (40–43) 1
56  0
18 95
8  16
0

5
9 (4
6–7
5) 42 (40–44) 2
09  0
45 101
3  15
9

0
581 < 0
001 < 0
001 < 0
001

ADMA (lM)

0
46  0
06

0
45  0
05

0
44  0
06

0
46  0
06

0
970

SDMA (lM)

0
51 (0
45–0
55)

0
49 (0
44–0
56)

0
48 (0
43–0
54)

0
48 (0
42–0
55)

0
003

Prior cardiovascular disease (%)

53
5

43
1

47
3

49
6

0
692

Ex- and active smokers (%)

65
6

63
2

56
7

57
7

0
092

Physical activity (h/day)

2
6 (1
0–4
2)

2
6 (1
3–3
9)

2
9 (1
6–4
7)

3
0 (1
5–4
4)

0
302

Antihypertensive drugs (%)

37
1

32
9

40
7

38
3

0
522

Oral antidiabetic drugs (%)

10
6

11
8

5
3

11
4

0
703

Lipid-lowering drugs (%)

16
6

11
2

13
3

23
5

0
089

BP, blood pressure; GFR, glomerular filtration rate; BNP, B-type natriuretic peptide; PTH, parathyroid hormone; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethlyarginine. Continuous data are presented as means  SD or as medians (with interquartile ranges); categorical data are presented as percentages. Data are analysed by ANOVA and chi-square tests with P for trend. *Available in 502 study participants.

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Table 2 Hazard ratios for mortality according to sex-specific homoarginine quartiles

Homoarginine (lM) Number of participants Number of deaths

1st quartile

2nd quartile

3rd quartile

0
46–1
27

1
01–1
55

1
26–1
90

152

153

47 (30
9%)

Adjustments

19 (12
4%)

151 20 (13
2%)

4th quartile 1
55–4
34 150 26 (17
3%)

All-cause mortality hazard ratios (with 95% confidence intervals)

Crude

2
01 (1
25–3
26)

0
70 (0
39–1
27)

0
76 (0
43–1
36)

1
00 reference

Model 1 = age and sex

1
78 (1
10–2
88)

0
69 (0
38–1
24)

0
77 (0
43–1
38)

1
00 reference

Model 2 = age, sex and BMI

1
87 (1
14–3
05)

0
70 (0
39–1
28)

0
79 (0
44–1
41)

1
00 reference

Model 2 + albumin and CRP

1
73 (1
05–2
84)

0
67 (0
37–1
21)

0
76 (0
42–1
36)

1
00 reference

Model 2 + GFR

1
99 (1
21–3
25)

0
71 (0
39–1
29)

0
83 (0
46–1
49)

1
00 reference

Model 2 + HbA1c and systolic BP

2
10 (1
27–3
47)

0
69 (0
38–1
27)

0
93 (0
51–1
68)

1
00 reference

Model 2 + lipids*

1
81 (1
11–2
97)

0
70 (0
39–1
28)

0
79 (0
44–1
42)

1
00 reference

†

Model 2 + drugs

1
98 (1
21–3
25)

0
77 (0
42–1
40)

0
85 (0
47–1
53)

1
00 reference

Model 2 + physical activity

2
02 (1
23–3
33)

0
74 (0
40–1
34)

0
84 (0
47–1
52)

1
00 reference

Model 2 + PTH and 25(OH)D

2
40 (1
39–4
15)

0
86 (0
44–1
66)

0
91 (0
48–1
75)

1
00 reference

Model 2 + ex- and active smokers

2
00 (1
22–3
28)

0
73 (0
40–1
32)

0
84 (0
46–1
51)

1
00 reference

Model 2 + microalbuminuria

1
89 (1
15–3
12)

0
71 (0
39–1
31)

0
81 (0
45–1
46)

1
00 reference

Model 2 + arginine

1
81 (1
09–3
03)

0
70 (0
38–1
27)

0
78 (0
44–1
40)

1
00 reference

Model 2 + ADMA and SDMA

1
85 (1
14–3
03)

0
72 (0
39–1
31)

0
80 (0
44–1
44)

1
00 reference

Model 2 + cardiovascular disease

2
02 (1
20–3
38)

0
76 (0
41–1
41)

0
81 (0
44–1
49)

1
00 reference

1
78 (1
03–3
06)

0
83 (0
44–1
57)

0
83 (0
44–1
58)

1
00 reference

‡

Model 2 + BNP

BMI, body mass index; CRP, C-reactive protein; GFR, glomerular filtration rate; BP, blood pressure; PTH, parathyroid hormone; 25(OH)D, 25-hydroxyvitamin D; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; BNP, B-type natriuretic peptide. *LDL cholesterol, HDL cholesterol, triglycerides. † Antihypertensive drugs, lipid-lowering drugs and oral antidiabetic drugs. ‡ Available in 502 study participants.

Discussion In this population-based cohort of older men and women, we observed that low homoarginine plasma concentrations were significantly associated with all-cause mortality and in particular with cardiovascular mortality. These results remained significant even after adjustments for various possible confounders. Our results are in line with the previous findings in female nursing home residents, in chronic kidney disease, heart failure and stroke patients, as well as in patients undergoing coronary angiography [2–7,27,28]. We want to emphasise that our present work was urgently needed, to clarify whether the associations between homoarginine deficiency and mortality are only evident in diseased populations or can also be observed in well characterised population-based cohorts such as the Hoorn

204

study. The fact that low homoarginine levels are strongly related to cardiovascular mortality, and not to cancer mortality, suggests that homoarginine deficiency is not a nonspecific indicator of adverse health outcomes, but is particularly associated with cardiovascular outcomes. Besides confirming the significant associations between homoarginine levels and fatal events, we attempted to elucidate whether associations with conventional and emerging cardiovascular risk factors might underlie these findings. Positive correlations of homoarginine with BMI, serum albumin, glucose levels and arginine might indeed suggest that homoarginine deficiency may indicate a wasting process. Associations between homoarginine levels and outcome were however materially unchanged after adjustments for these variables. Moreover, the non-existing association between arginine levels and any mortality outcome argues against the hypothesis that

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HOMOARGININE AND MORTALITY IN AN OLDER POPULATION

Table 3 Hazard ratios for all-cause, cardiovascular and cancer mortality according to sex-specific homoarginine quartiles All-cause mortality Cardiovascular mortality Subjects at risk/events (% of events among subjects at risk)

Cancer mortality

606/112 (18
5)

581/31 (5
3)

581/30 (5
2)

First homoarginine quartile

152/47 (30
9)

142/18 (12
7)

142/8 (5
6)

Upper three homoarginine quartiles

454/65 (14
3)

439/13 (3
0)

439/22 (5
0)

Entire cohort

Adjustments

Hazard ratios (with 95% confidence intervals) in the first compared to the upper three sex-specific homoarginine quartiles

Crude

2
47 (1
69–3
59)

4
76 (2
33–9
72)

1
27 (0
56–2
84)

Model 1 = age and sex

2
18 (1
50–3
19)

4
02 (1
96–8
25)

1
25 (0
55–2
81)

Model 2 = age, sex and BMI

2
26 (1
54–3
32)

4
20 (2
03–8
69)

1
25 (0
55–2
85)

Model 2 + albumin and CRP

2
17 (1
47–3
19)

3
95 (1
89–8
27)

1
17 (0
51–2
69)

Model 2 + GFR

2
36 (1
61–3
46)

5
12 (2
45–10
69)

1
23 (0
54–2
81)

Model 2 + HbA1c and systolic BP

2
43 (1
64–3
59)

4
35 (2
08–9
11)

1
28 (0
56–2
92)

Model 2 + lipids*

2
19 (1
48–3
23)

3
91 (1
84–8
28)

1
29 (0
57–2
93)

Model 2 + drugs†

2
27 (1
55–3
34)

4
19 (2
01–8
77)

1
26 (0
55–2
87)

Model 2 + physical activity

2
37 (1
61–3
48)

4
29 (2
07–8
89)

1
26 (0
56–2
87)

Model 2 + PTH and 25(OH)D

2
60 (1
74–3
90)

4
55 (2
07–9
99)

1
82 (0
76–4
35)

Model 2 + ex- and active smokers

2
36 (1
61–3
45)

4
38 (2
13–9
02)

1
25 (0
55–2
83)

Model 2 + microalbuminuria

2
27 (1
54–3
33)

4
22 (2
03–8
75)

1
26 (0
55–2
86)

Model 2 + arginine

2
25 (1
50–3
33)

3
90 (1
85–8
22)

1
34 (0
56–3
16)

Model 2 + ADMA and SDMA

2
21 (1
51–3
24)

4
24 (2
02–8
90)

1
28 (0
56–2
91)

Model 2 + cardiovascular disease

2
37 (1
59–3
52)

4
24 (1
92–9
36)

1
38 (0
60–3
18)

2
00 (1
31–3
06)

3
75 (1
57–9
00)

1
00 (0
40–2
50)

‡

Model 2 + BNP

BMI, body mass index; CRP, C-reactive protein; GFR, glomerular filtration rate; BP, blood pressure; PTH, parathyroid hormone; 25(OH)D, 25-hydroxyvitamin D; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; BNP, B-type natriuretic peptide. *LDL cholesterol, HDL cholesterol, triglycerides. † Antihypertensive drugs, lipid-lowering drugs and oral antidiabetic drugs. ‡ Available in 502 study participants.

arginine may underlie the homoarginine findings. Nevertheless, the significant relationship between homoarginine and arginine might suggest that homoarginine levels reflect nutritional intake or gastrointestinal uptake of amino acids. In this context, it should also be stressed that lysine is considered to serve as the main precursor for homoarginine synthesis by AGAT, which produces homoarginine by transferring an amidino group from arginine to lysine [9,15,16]. Although previous studies suggest that the kidney is the major site for endogenous homoarginine production and that lower homoarginine levels are associated with the prevalence and progression of chronic kidney disease, we did not observe a significant association of homoarginine and GFR [29]. SDMA, another marker of renal dysfunction and adverse outcomes, was however inversely related to homoarginine in our study

[30]. As for other mortality risk factors, SDMA adjustments had no significant effect on the relationship between homoarginine and mortality. Further studies covering a broader range of chronic kidney disease stages are therefore warranted to elucidate the associations of homoarginine with renal dysfunction. In previous cross-sectional analyses of the Hoorn study, we have already reported that homoarginine was positively associated with blood pressure [20]. This finding confirms the data from patients referred to coronary angiography [4]. In line with this, experiments in rats showed that homoarginine infusions decreased renal medullary blood flow and NO levels [31]. In contrast, another study in rats showed that homoarginine supplementation exerted antihypertensive effects, which were accompanied with increased excretion of nitrate, the degradation product of NO [11]. Moreover, it has been shown that low

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Figure 1 Kaplan–Meier curves for all-cause mortality for the first vs. the upper three sex-specific homoarginine quartiles (log-rank test: P < 0
001).

Figure 2 Kaplan–Meier curves for cardiovascular mortality for the first vs. the upper three sex-specific homoarginine quartiles (log-rank test: P < 0
001).

plasma concentrations of homoarginine indicate an increased risk of early pre-eclampsia, which is characterised by elevated blood pressure and proteinuria [32]. We therefore have to acknowledge that the impact of homoarginine on the availability of NO and its subsequent effect on blood pressure and endothelial function is still conflicting at present [9–12,32]. Further in-depth studies, including more sophisticated measurements of blood pressure (i.e. 24-h ambulatory blood pressure measurements) and simultaneous determinations of homoarginine and NO levels or parameters which indicate NO availability and endothelial dysfunction, are therefore warranted.

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Another important finding was the significant inverse association between homoarginine and BNP, suggesting that homoarginine deficiency is related to myocardial dysfunction. This confirms the previous investigations showing that low homoarginine is significantly associated with heart failure in diabetic dialysis patients and patients referred for coronary angiography [4,5]. Although adjustments for BNP did not significantly attenuate the association between homoarginine and fatal events, it is obvious from our and previous studies that low homoarginine indicates heart diseases. Activity of AGAT, the key enzyme for homoarginine synthesis, may underlie the association between homoarginine and heart function, because AGAT is also crucial for energy metabolism by catalysing the production of GAA. In this context, it should also be underlined that although AGAT is considered the main enzyme for homoarginine formation, it catalyses various reactions with the main function to synthesise GAA by transferring an amidino group from arginine to glycine [15]. GAA is further converted to creatine, which functions as an energy shuttle [33]. It has therefore been hypothesised that previously observed upregulation of AGAT in the failing heart and in muscles of mice with Duchennes muscular dystrophy aims to compensate for energy deficits in these diseases [19,34]. This hypothesis fits well with the clinical presentation of the extremely rare disease of inherited AGAT deficiency, which is characterised by muscle weakness, low body weight and mental retardation [35,36]. Patients with AGAT deficiency can be effectively treated with oral creatine, supporting the role of AGAT in energy metabolism [35,36]. Detailed characterisation of glucose metabolism is missing in these AGAT-deficient patients, but it has been shown that AGAT deficiency in mice protects from metabolic syndrome [37]. Assuming that low-serum homoarginine indicates reduced AGAT activity, data from AGAT-deficient mice on attenuated gluconeogenesis and enhanced glucose tolerance are in line with our findings on significantly reduced glucose levels in participants with low homoarginine [37]. Enhanced activity of AMP-activated protein kinase (AMPK) in AGAT deficiency was described as the mechanism leading to reduced blood glucose levels, because AMPK activation increases glucose uptake into skeletal muscle and inhibits gluconeogenesis [37]. Considering these interesting findings, further studies are required to evaluate in more detail the relationship between homoarginine, AGAT activity and glucose metabolism. Additional studies including homoarginine supplementation will also be needed to clarify whether homoarginine has some clinically relevant effects in humans or whether homoarginine is just a marker for pathophysiological processes such as AGAT activity. Regarding our mortality results, we also want to draw attention to the fact that homoarginine has been used as an internal standard for laboratory measurements, such as ADMA [9,38,39]. Consequently, a lower endogenous homoarginine concentration

ª 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd

HOMOARGININE AND MORTALITY IN AN OLDER POPULATION

might have contributed to higher ADMA values in these reports [8,38,39]. Our findings therefore strongly argue against using homoarginine as an internal standard in laboratory procedures, and caution is warranted when interpreting the results of ADMA levels measured by such methods [9,38,39]. Our results are limited because we studied a cohort of older men and women in the Netherlands, and our results may therefore not be generalisable to other study cohorts or a younger general population. Another limitation of our work is the lack of dietary data and the relatively low number of fatal events, in particular cardiovascular events. Due to the observational nature of our study, we cannot draw definite conclusions regarding causality for the associations between homoarginine and mortality. Although our results remained significant despite adjustments for various possible confounders, such as markers of malnutrition, GFR or BNP, we cannot rule out the existence of unmeasured or unknown confounding factors. Our results are also limited by the fact that we did not perform detailed determinations of other relevant amino acids, energy metabolites and activity or expression of AGAT and its associations with homoarginine levels. Such measurements will be important in future studies to shed more light on the pathophysiology of homoarginine metabolism. It is at present premature to advocate homoarginine supplementation to prevent adverse health consequences, but our data point to the need for further experimental and clinical studies to evaluate whether homoarginine supplementation or the modulation of homoarginine metabolism (e.g. AGAT activity) could have beneficial effects on overall and cardiovascular health. In summary, we observed that low homoarginine concentrations are a risk marker for all-cause mortality and especially of cardiovascular mortality in older women and men. Further studies are urgently needed to confirm our findings and to elucidate the underlying pathophysiological mechanisms.

Centre, van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands (S. Pilz, F. Rutters, J. M. Dekker); Department of Internal Medicine, Division of Endocrinology and Metabolism, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria (S. Pilz, K. Kienreich); Metabolic Laboratory, Department of Clinical Chemistry and Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, De Boelelaan 1117, PO Box 7057, 1007 MB Amsterdam, the Netherlands (T. Teerlink, P. G. Scheffer); Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria (A. Meinitzer, W. M€ arz); Department of Internal Medicine, Division of Cardiology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria (A. Tomaschitz); Division of Nephrology, Department of Medicine, University of Wu¨rzburg, Oberduerrbacherstr 6, 97080 Wu¨rzburg, Germany (C. Drechsler); Department of General Practice and the EMGO Institute for Health and Care Research, VU University Medical Centre, van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands (G. Nijpels); Department of Internal Medicine and Cardiovascular Research Institute Maastricht, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands (C. D. A. Stehouwer); Department of Medicine V (Nephrology, Hypertensiology, Endocrinology, Diabetology, Rheumatology), Medical Faculty Mannheim, University of Heidelberg, 69115 Mannheim, Germany (W. M€ arz); Synlab Academy, Synlab Services GmbH, Gottlieb Daimler Strasse 25, 68165 Mannheim, Germany (W. M€ arz).

Acknowledgements

Received 25 May 2013; accepted 15 November 2013

The authors thank all the study participants and all the individuals who supported and contributed to the Hoorn study. The authors thank Sigrid de Jong for performing the analyses of homoarginine and related amino acids.

Contributions All authors contributed to the drafting and supervision of the manuscript. SP, JMD and FR performed the statistical analyses. TT, PS, GN, CDAS and JMD designed the study and collected the data. All authors have critically reviewed and revised the manuscript. None of the authors has conflict of interests. Address Department of Epidemiology and Biostatistics, EMGO Institute for Health and Care Research, VU University Medical

Correspondence to: Stefan Pilz, MD, PhD, Department of Internal Medicine, Division of Endocrinology and Metabolism, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria. Tel.: +43 650 9103667; fax: +43 316 673216; e-mail: [email protected]

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