The major clinical consequence of type 2 diabetes is mortality and morbidity from atherosclerotic vascular disease. The degree of atherosclerosis can be evaluated by high-resolution B-mode ultrasound scan (1). Using high-resolution B-mode ultrasound scan, we recently demonstrated that serum nonesterified fatty acids (NEFAs) are independently associated with carotid atherosclerosis in nonobese nonhypertensive Japanese type 2 diabetic patients (2). Michaud et al. (3) recently reported that fatty acids enhance lipoprotein lipase (LPL) production in human macrophages. LPL secreted from macrophages is known to contribute to the development and progression of atherosclerosis (4). Thus, fatty acids are considered to be associated with atherosclerotic vascular disease in type 2 diabetic patients.

Slightly increased urinary albumin excretion rate, microalbuminuria, is considered as an index of diabetic nephropathy. Microalbuminuria has also been identified as an independent risk factor for atherosclerosis in type 2 diabetic patients. An association between microalbuminuria and cardiac disease in type 2 diabetic patients has been demonstrated (5). To the best of our knowledge, however, it remains to be clarified whether type 2 diabetic patients with microalbuminuria have higher concentrations of serum NEFAs than those with normoalbuminuria. In this context, a major problem is that microalbuminuria has been associated with atherosclerotic risk factors, such as high blood pressure, hypertriglyceridemia, and low HDL cholesterol, thus complicating the relation of microalbuminiuria with serum NEFAs (6,7). Moreover, it is well recognized that being overweight or hyperglycemic per se affects microalbuminuria and serum NEFA levels in humans. We therefore recruited nonobese type 2 diabetic patients with microalbuminuria who were carefully matched for BMI, blood pressure, serum lipid level, and fasting glucose to those with normoalbuminuria. This is the first description that serum NEFAs are increased in nonobese Japanese type 2 diabetic patients with microalbuminuria.

A total of 21 diabetic patients with microalbuminuria (15 men and 6 women) and 45 patients with normoalbuminuria (33 men and 12 women) were enrolled in the study. They all were nonobese (BMI <27 kg/m2) Japanese type 2 diabetic patients (8). Type 2 diabetes was diagnosed based on the criteria of the World Health Organization (9). All subjects had ingested at least 150 g carbohydrate for the 3 days preceding the study. A total of 14 (67%) patients with microalbuminuria and 23 (51%) patients with normoalbuminuria were taking sulfonylureas. The rest were treated with diet alone. None had received insulin therapy. Ten (48%) patients with microalbuminuria and 15 (33%) patients with normoalbuminuria were treated with antihypertensive drugs. Four (19%) patients with microalbuminuria and seven (16%) patients with normoalbuminuria were treated with lipid-lowering agents. There was no significant difference in sex and medication status between the patients with microalbuminuria and those with normoalbuminuria. They did not consume alcohol or perform heavy exercise for at least 1 week before the study.

Blood was drawn on the morning after a 12-h fast. Plasma glucose was measured with the glucose oxidase method. Total, HDL, and LDL cholesterol, triglycerides, remnant-like particle cholesterol (RLP-C), and NEFAs were also measured. The LDL cholesterol level was calculated using the Friedewald formula (10). RLP-C level was measured by the method reported by Nakajima et al. (11). Serum NEFAs were measured in duplicate using the enzymatic method (NEFA HR kit; Wako Chemicals, Osaka, Japan), and the mean of the two values was used (2). The coefficient of variation for NEFAs was 2%.

Urinary albumin concentration was assessed in a morning spot urine sample using the single radial immunodiffusion method (12). Urinary albumin concentration was measured in duplicate, and the mean of the two values was used for the study. Intra- and interassay coefficients of variations were <6% (Alb-Tia Seiken; Denka-Seiken, Tokyo, Japan). Several reports have indicated that early morning spot urine is usually sufficient for detecting the presence of microalbuminuria (13,14). In the present study, we calculated urinary albumin excretion rate as a ratio of urinary albumin and urinary creatinine, a ratio that markedly enhances the accuracy of the single spot urine sample in the assessment of microalbuminuria (15). Microalbumiuria was defined as a urinary albumin concentration >30 but <300 mg/g creatinine. Normoalbuminuria was defined as urinary albumin concentration <30 mg/g creatinine.

The statistical analysis was performed with the StatView 5.0 system (Statview, Berkeley, CA). The differences of mean were determined by Student’s t test. Data were expressed as the means ± SEM.

The clinical characteristics and profile between the patients with microalbuminuria (n = 21) and normoalbuminuria (n = 45) were compared. Urinary albumin concentrations in the patients with microalbuminuria and normoalbuminuria were 89 ± 12 mg/g creatinine (range 44–236) and 5 ± 1 (range 0.1–17.0), respectively. No significant difference was observed in age (64.2 ± 2.2 vs. 61.8 ± 1.0 years, P = 0.127), BMI (22.7 ± 0.4 vs. 22.8 ± 0.3 kg/m2, P = 0.481), or systolic (128 ± 2 vs. 126 ± 2 mmHg, P = 0.288) and diastolic (73 ± 2 vs. 73 ± 1 mmHg, P = 0.448) blood pressure between the two groups. Fasting glucose (154 ± 7 vs. 143 ± 5 mg/dl, P = 0.109) and HbA1c (7.5 ± 0.2 vs. 7.0 ± 0.2%, P = 0.066) levels were higher in patients with microalbuminuria than in those with normoalbuminuria, but the difference was not statistically significant. The patients with microalbuminuria had higher triglycerides (127 ± 13 vs. 110 ± 5 mg/dl, P = 0.066) and higher RLP-C (5.3 ± 0.5 vs. 4.9 ± 0.2 mg/dl, P = 0.208) concentrations than those with normoalbuminuria, but the difference was not statistically significant. There was no significant difference in serum total (200 ± 7 vs. 195 ± 5 mg/dl, P = 0.290), HDL (51 ± 3 vs. 50 ± 2 mg/dl, P = 0.279), and LDL cholesterol (124 ± 4 vs. 124 ± 6 mg/dl, P = 0.478) levels between the two groups. In contrast, serum NEFAs were significantly higher in patients with microalbuminuria (0.64 ± 0.05 mEq/l) than in those with normoalbuminuria (0.51 ± 0.02 mEq/l, P = 0.002).

In the present study, we first confirmed the presence of elevated NEFAs in nonobese Japanese type 2 diabetic patients with microalbuminuria. Microalbuminuria has been shown to be not only an indicator of incipient nephropathy, but also a risk marker for early mortality, especially in type 2 diabetes, due to cardiovascular diseases.

Although the mechanisms underlying the relation between NEFAs and microalbuminuria are unclear, serum NEFAs seem to be associated with early atherosclerotic changes in nonobese Japanese type 2 diabetic patients. Using high-resolution B-mode ultrasound scan, we very recently demonstrated that serum NEFAs are independently associated with carotid atherosclerosis in nonobese, nonhypertensive, well-controlled (mean HbA1c 7.0%), unique type 2 diabetic patients (2). Interestingly, the degree of carotid stenosis was 8.1 ± 2.1%, suggesting that serum NEFAs are associated with the very early stages of carotid atherosclerosis. Microalbuminuria is also considered to be a risk marker for the early events in athrosclerosis. Thus, it may be hypothesized that serum NEFA level is reflective of the early stage of atherosclerosis in nonobese Japanese type 2 diabetic patients.

The mechanism by which serum NEFAs are associated with atherosclerosis in nonobese Japanese type 2 diabetic patients is unclear. In this respect, the recent report of Michaud et al. (3) that fatty acids enhance macrophage LPL production is very interesting. LPL secreted from macrophage is known to contribute to the development and progression of atherosclerosis (4). Alternatively, serum NEFAs might cause atherosclerosis by stimulating a coagulation cascade sequence. Didisheim et al. (16) previously showed that saturated long-chain fatty acids activate Hageman factor (factor XII). Hageman factor initiates the cascade sequence of enzymatic reactions, culminating in the production of thrombin and the conversion of fibrinogen to fibrin. Thrombin also induces an increase in fibrinogen biosynthesis. Pilgeram and Pickart (17) have shown that free fatty acids (FFAs) stimulate the rate of biosynthesis of fibrinogen in vitro. Schneider et al. (18) demonstrated that FFAs had synergistic effects on the insulin-stimulated increase in plasminogen activator inhibitor-1 (PAI-1) in the blood of type 2 diabetic patients. Enhanced coagulation cascade sequence in conjunction with decreased fibrinolytic capacity due to overexpression of PAI-1 might explain the reason why serum NEFA level is associated with the early stage of atherosclerosis and microalbuminuria in nonobese Japanese type 2 diabetic patients.

In summary, we first demonstrated that serum NEFAs are increased in nonobese Japanese type 2 diabetic patients with microalbuminuria. Further study should be undertaken to clarify whether serum NEFAs are reflective of the early stages of atherosclerosis, including microalbuminuria, in nonobese Japanese type 2 diabetic patients.

1.
O’Leary DH, Polak JF, Wolfson SK Jr, Bond MG, Bommer W, Sheth S, Psaty BM, Sharrett AR, Manolio TA: Use of sonography to evaluate carotid atherosclerosis in the elderly: the Cardiovascular Health Study.
Stroke
22
:
1155
–1163,
1991
2.
Taniguchi A, Sakai M, Teramura S, Fukushima M, Hama K, Marumoto K, Nezumi N, Yoshida T, Nagasaka S, Hayashi R, Tokuyama K, Nakai Y: Serum nonesterified fatty acids are related with carotid atherosclerotic plaque in nonobese nonhypertensive Japanese type 2 diabetic patients (Letter).
Diabetes Care
24
:
1505
–1507,
2001
3.
Michaud SE, Renier G: Direct regulatory effect of fatty acids on macrophage lipoprotein lipase: potential role of PPARs.
Diabetes
50
:
660
–666,
2001
4.
Babaev VR, Fazio S, Gleaves LA, Carter KJ, Semenkovich CF, Linton MF: Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo.
J Clin Invest
103
:
1697
–1705,
1999
5.
Mogensen CE: Microalbuminuria predicts clinical proteinuria and early mortality in maturity-onset diabetes.
N Engl J Med
310
:
356
–360,
1984
6.
Haffner SM, Stern MP, Gruber MK, Hazuda HP, Mitchell BD, Patterson JK: Microalbuminuria: potential marker for increased cardiovascular risk factors in non-diabetic subjects?
Atherosclerosis
10
:
727
–731,
1990
7.
Mykkanen L, Haffner SM, Kuusisto J, Pyorala K, Laakso M: Microalbuminuria precedes the development of NIDDM.
Diabetes
43
:
552
–557,
1994
8.
Taniguchi A, Nakai Y, Doi K, Fukuzawa H, Fukushima M, Kawamura H, Tokuyama K, Suzuki M, Fujitani J, Tanaka H, Nagata I: Insulin sensitivity, insulin secretion, and glucose effectiveness in obese subjects: a minimal model analysis.
Metabolism
44
:
1397
–1400,
1995
9.
World Health Organization: Diabetes Mellitus: Report of a WHO Study Group. Geneva, World Health Org., 1985 (Tech. Rep. Ser., no. 727)
10.
Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.
Clin Chem
18
:
499
–502,
1972
11.
Nakajima K, Saito T, Tamura A, Suzuki M, Nakano T, Adachi M, Tanaka A, Tada N, Nakamura H, Campos EE, Havel RJ: Cholseterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gels.
Clin Chim Acta
223
:
53
–71,
1993
12.
Becker W: Determination of antisera titres using the single radial immunodiffusion method.
Immunochem
6
:
539
–541,
1969
13.
Gatling W, Knight C, Hill RD: Screening for early diabetic nephropathy: which sample to detect microalbuminuria?
Diabet Med
2
:
451
–455,
1985
14.
Cowell CT, Rodgers S, Silkink M: First morning urinary albumin concentration is a good predictor of 24-hour urinary albumin excretion in children with type 1 (insulin-dependent) diabetes.
Diabetologia
29
:
97
–99,
1986
15.
Hutchison AS, O’Reilly DSJ, McCuish AC: Albumin excretion rate, albumin concentration, and albumin/creatinine ratio compared for screening diabetics for slight albuminuria.
Clin Chem
34
:
2019
–2021,
1988
16.
Didisheim P, Mibashan RS: Activation of Hageman factor (factor XII) by long-chain saturated fatty acids.
Thromb Diath Haemorrh
9
:
346
–353,
1963
17.
Pilgeram LO, Pickart LR: Control of fibrinogen biosynthesis: the role of free fatty acid.
J Athero Res
8
:
155
–166,
1968
18.
Schneider DJ, Sobel BE: Synergistic augmentation of expression of plasminogen activator inhibitor type-1 induced by insulin, very-low-density lipoproteins, and fatty acids.
Coronary Artery Dis
7
:
813
–817,
1996

Address correspondence to Ataru Taniguchi, MD, First Department of Internal Medicine, Kansai-Denryoku Hospital, 2-1-7 Fukushima, Fukushima-ku, Osaka-city, Osaka 553-0003 Japan. E-mail: K-58403@kepco.co.jp.