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Journal of Diabetes and Its Complications 20 (2006) 133–135

Letters to the Editor

How to investigate skin endothelial dysfunction

in diabetes

To the Editor,

Microvascular dysfunction is a keystone of diabetes

complications, characterized by an abnormal structure and

function. Microvascular dysfunction progresses in silence

for years before clinical disease occurs, but it may preexist

at the time of diagnosis. However, conflicting data exist

concerning the effect of acute hyperglycemia on vascular

function in humans. Among the different organs where

microvascular dysfunctions predominantly occur, eyes and

skin appear to be the most accessible. This has led to the

development of specific tools, such as laser Doppler

flowmetry, which enables easy, noninvasive measurement

of skin blood flow. In a recent study, Forst et al. (2005)

suggested that postprandial microvascular function and

endothelial cell function were disturbed in patients with

type I diabetes, and that improvement of postprandial

metabolic control improved it. Although the study is of

interest, both the use of acetylcholine iontophoresis as a

marker of skin endothelial function and the use of basal

skin flow measurements expressed as absolute value

deserve criticism.

The endothelium is one of the key vascular wall layers

that are affected during the early stages of diabetes and is

characterized by decreased NO availability. Furchgott and

Zawadzki (1980) used acetylcholine to demonstrate that the

endothelium of conductance arteries was able to dilate vas-

cular beds through the release of what was later established

to be NO. Subsequently, acetylcholine has commonly been

used as a test for NO release through the endothelium.

However, several pertinent limitations prevent its extrap-

olation in human skin microcirculation. First, acetylcholine

iontophoresis is far from being a gold standard for

endothelial function in human skin, as suggested by the

title of the paper. In a recent study where acetylcholine was

infused through a subdermal microdialysis fiber, it was

clearly shown that acetylcholine-mediated dilation of

human skin was unchanged following NO synthase

inhibition (Holowatz, Thompson, Minson, & Kenney,

2005), further confirming previous in vitro studies (Buus,

Simonsen, Pilegaard, & Mulvany, 2000). Indeed, a large

part of the response seems to be mediated by prostanoids.

Furthermore, acetylcholine iontophoresis induces an axon

– see front matter D 2006 Elsevier Inc. All rights reserved.

reflex that participates to the increase in skin cutaneous

blood flow (Berghoff, Kathpal, Kilo, Hilz, & Freeman,

2002), as demonstrated by the lower increase in skin blood

flow at sites under local anesthesia. In addition, anodal

current on its own causes a current induced hyperhemia

(Durand, Fromy, Bouye, Saumet, & Abraham, 2002).

Therefore, the assumption that acetylcholine iontophoresis

is a functional marker for endothelial function is debatable.

Second, laser Doppler flowmetry remains a semiquantita-

tive approach to investigate skin blood flow (Carpentier,

1999). Using a single probe, the intra- and interindividual

coefficients of variation of the basal skin blood flow are

higher than the differences observed in the postprandial state

in the present study (Bircher, de Boer, Agner, Wahlberg, &

Serup, 1994). In addition, expression of data in terms of

perfusion units does not take into consideration potential

variations of blood pressure, which could alter micro-

circulatory flow. Thus, cutaneous vascular conductance

rather than flux would be a better physiological index to

study vasodilation. Furthermore, given the semiquantitative

approach of laser Doppler measurements, baseline mea-

surements- and acetylcholine-induced vasodilation would

best be expressed as a percentage of a maximal vasodilation

such as high concentrations of nitrates or local heating. This

would avoid a bias induced by the heterogeneity of skin

cutaneous blood flow from site to site. If this cannot be

performed, a non-endothelial-dependent relaxation should

be performed and compared to the effect of acetylcholine.

This minimizes the high individual variability and provides

support for endothelial vs. structural (non-endothelial-

dependent) vasodilation.

Investigation of skin blood flow using laser Doppler

flowmetry is an easy, noninvasive approach that can be

coupled with acetylcholine iontophoresis. However, this

methodology exhibits many pitfalls that, at this time, prevent

its widespread use in clinical settings as a functional marker

of skin endothelial function.

Jean-Luc Cracowski

Gregg R. McCord

Christopher T. Minson

Department of Human Physiology

122 C Esslinger Hall

1240 University of Oregon

Eugene, OR 97403-1240, USA

Letters to the Editor / Journal of Diabetes and Its Complications 20 (2006) 133–135134

Tel.: +1 541 346 4105

Fax: +1 541 346 2841

E-mail address: jean-luc.cracowski@ujf-grenoble.fr

doi:10.1016/j.jdiacomp.2005.10.002

References

Berghoff, M., Kathpal, M., Kilo, S., Hilz, M. J., & Freeman, R. (2002).

Vascular and neural mechanisms of ACh-mediated vasodilation in the

forearm cutaneous microcirculation. Journal of Applied Physiology, 92,

780–788.

Bircher, A., de Boer, E. M., Agner, T., Wahlberg, J. E., & Serup, J. (1994).

Guidelines for measurement of cutaneous blood flow by laser Doppler

flowmetry. A report from the Standardization Group of the European

Society of Contact Dermatitis. Contact Dermatitis, 30, 65–72.

Buus, N. H., Simonsen, U., Pilegaard, H. K., & Mulvany, M. J. (2000).

Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in

acetylcholine relaxation of isolated human small arteries. British

Journal of Pharmacology, 129, 184–192.

Carpentier, P. H. (1999). New techniques for clinical assessment of the

peripheral microcirculation. Drugs, 59 Spec No, 17–22.

Durand, S., Fromy, B., Bouye, P., Saumet, J. L., & Abraham, P. (2002).

Vasodilatation in response to repeated anodal current application in the

human skin relies on aspirin-sensitive mechanisms. Journal of

Physiology, 540, 261–269.

Forst, T., Forst, S., Strunk, K., Lobig, M., Welter, K., Kazda, C., &

Pfutzner, A. (2005). Impact of insulin on microvascular blood flow and

endothelial cell function in the postprandial state in patients with Type 1

diabetes. Journal of Diabetes and its Complications, 19, 128–132.

Furchgott, R. F., & Zawadzki, J. V. (1980). The obligatory role of

endothelial cells in the relaxation of arterial smooth muscle by

acetylcholine. Nature, 288, 373–376.

Holowatz, L. A., Thompson, C. S., Minson, C. T., & Kenney, W. L. (2005).

Mechanisms of acetylcholine-mediated vasodilatation in young and

aged human skin. Journal of Physiology, 563, 965–973.

Response to Jean-Luc Cracowski, Gregg R. McCord,

Christopher T. Minson

To the Editor,

We agree that microvascular function is a major

contributor to the development of diabetic complications

and that it may preexist at the time of diagnosis of the

metabolic disease. We also agree that conflicting results

exist on the impact of hyperglycaemia on microvascular

blood flow in nondiabetic and diabetic patients. In the

postprandial state, several different mediators are thought

to independently interact with endothelial function and the

regulation of vascular blood flow (Forst, Kunt, et al., 1998;

Forst et al., 2005). While increasing glucose levels were

found to impair endothelial function and to reduce vascular

blood flow, increasing blood insulin and C-peptide levels

are thought to partially counteract these acute glucotoxic

effects in the postprandial situation (Ceriello et al., 2004;

Forst & Kunt, 2004; Williams et al., 1998). In patients

with diabetes mellitus, several of these vasoregulatory

mechanisms are more or less affected, which result in a

severely impaired postprandial microcirculation in several

tissues. The aim of our study was to investigate the effect

of the different pharmacokinetic properties of insulin lispro

and human regular insulin on postprandial dynamics of

microvascular blood flow regulation by the use of laser

Doppler fluxmetry.

The main finding of our study was that the postprandial

regulation of microvascular blood flow after insulin lispro

was much closer to the microvascular blood flow

regulation in nondiabetic control subjects compared with

the injection of regular insulin. In addition, it was found

that the postprandial microvascular response to acetylcho-

line was more pronounced after lispro injection compared

with regular insulin.

Iontophoresis of acetylcholine through the skin induces

microvascular hyperaemia by a complex activation of endo-

thelial and non-endothelium-dependent mechanisms (Forst,

Pfutzner, et al., 1998; Morris, Shore, & Tooke, 1995).

Transdermal application of acetylcholine induces vasodila-

tation and hyperaemia by the activation of an antidromic

axon reflex arc (Parkhouse&LeQuesne, 1988; Pfutzner et al.,

1999) and the release of vasodilators like nitric oxide,

prostacyclin, and endothelium-derived hyperpolarizing

factor from endothelial cells (Furchgott & Zawadski, 1988;

Panza, Quyyumi, Brush, & Epstein, 1990). Both acetylcho-

line-activated mechanisms are interrelated, and even

the neuronal vasodilatation is partly mediated by an NO-

dependentmechanism (Minson, Berry, & Joyner, 2001; Vinik

et al., 2001). It is still contradictory which endothelial

mediator of acetylcholine plays the most important role in

humans (Morris & Shore, 1996; Noon, Walker, Hand, &

Webb, 1998). In contrast to the investigation of Holowatz,

Thompson, Minson, and Kenney (2005), the acetylcholine

response was found to be NO mediated by Boutsiouki,

Georgiou, and Clough (2004) using the same methodology.

It was not our intention to interpret iontophoresis of

acetylcholine as the gold standard for the measurement of

endothelial function in the skin as it was misconceived by

Cracowski et al. Nevertheless, it is an easy and noninvasive

method for the investigation of microvascular blood flow

especially for longer observation periods and repeated

measurements. We agree the interindividual and even the

intraindividual coefficient of variation are high due to the

change in the position of the laser Doppler probe from

patient to patient or from day to day. In our study, the

position of the probe was kept constant for the whole

investigation resulting in pretty constant laser Doppler

readings, as published previously.

In particular, the different insulin formulations were

compared according to a crossover design with a randomised

sequence of regular insulin or insulin lispro injections.

Therefore, the differences between insulin lispro and regular

insulin in diabetic patients compared with nondiabetic

control subjects could not at all be explained by a high

coefficient of variation in laser-Doppler-flux measurements.