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Rheumatology 2008 47(1):80-83; doi:10.1093/rheumatology/kem300
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cold stimulus fingertip lacticemy test—an effective method to monitor acute therapeutic intervention on primary Raynaud's phenomenon and systemic sclerosis

S. M. A. Fontenelle, C. Kayser, M. L. C. Pucinelli and L. E. C. Andrade

Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.

Correspondence to: L. E. C. Andrade, Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 740, 3° andar, São Paulo, SP 04023-062, Brazil. E-mail: luis{at}reumato.epm.br


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. The recently developed cold stimulus fingertip lacticemy test (CS-FTL) provides biochemical assessment of peripheral perfusion in patients with Raynaud's phenomenon (RP). We evaluated how the CS-FTL test can assess the acute effect of nifedipine in microvascular dynamics on primary RP and RP secondary to SSc.

Methods. A double-blinded controlled trial with crossover design was performed in 20 primary RP and 20 SSc patients. Patients received one single sublingual placebo or 10 mg nifedipine capsule, with crossover after a 15-day washout period. FTL was determined in resting conditions (pre-CS-FTL) and 10 min after CS (post-CS-FTL), before and 1 h after drug administration. Percent variation in post- vs pre-CS-FTL was expressed as {Delta}CS-FTL.

Results. Before intervention, CS induced FTL decrease in primary RP ({Delta}CS-FTL = –21.3 ± 13.0%) and FTL increase in SSc patients ({Delta}CS-FTL = +24.5 ± 21.2%). Placebo had no effect on pre-CS-FTL, post-CS-FTL and {triangleup}CS-FTL in primary RP and SSc. Nifedipine induced a significant decrease in pre-CS-FTL (1.94 ± 0.45 vs 1.57 ± 0.41 mg/dl; P = 0.005) and post-CS-FTL (1.53 ± 0.35 vs 1.32 ± 0.37 mg/dl; P = 0.004) in primary RP and a significant decrease in post-CS-FTL (3.18 ± 1.43 vs 2.56 ± 1.30 mg/dl; P = 0.028) and {Delta}CS-FTL (+15.9 ± 24.7% vs –12.9 ± 16.6%; P = 0.001) in SSc.

Conclusions. The CS-FTL test was able to demonstrate and quantify a dual effect of nifedipine on the biochemical dimension of peripheral perfusion in primary RP and in SSc patients in which there was a significant improvement in tissue perfusion in resting conditions and after exposure to a CS. The CS-FTL test will enrich the armamentarium for investigation and clinical evaluation of conditions associated with RP.

KEY WORDS: Raynaud's phenomenon, Systemic sclerosis, Lactate, Microcirculation, Calcium channel blockers


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Raynaud's phenomenon (RP) affects 5–15% of the general population and is a frequent manifestation in autoimmune rheumatic diseases, especially those related to the SSc spectrum [1, 2]. Calcium-channel blockers, particularly nifedipine, are widely used in the treatment of RP. Nifedipine is a vasodilator of arteries and arterioles that inhibits intracellular calcium influx and calcium-dependent vascular smooth muscle contraction [3, 4]. Controlled clinical trials have shown that nifedipine induces an increase in peripheral blood flow and a significant decrease in the frequency and severity of the ischaemic attacks in RP patients [5–8]. The beneficial effects of acute nifedipine on RP have been documented by plethysmography [6, 9], computer-aided fluorescence video-microscopy [10], measurement of finger temperature and laser Doppler skin blood flow [11]. However, there is no objective evidence that the improvement in the clinical aspects and in flow parameters is necessarily associated with improvement in biochemical parameters indicating more effective tissue perfusion in RP.

The determination of lactic acid concentration in blood obtained from fingertips [fingertip lacticemy (FTL)] before and after a cold stimulus (CS) is a recently developed technique to evaluate the degree of local anaerobiosis during the ischaemic stage of RP [12]. The CS-FTL has been standardized in normal individuals and its behaviour has been documented in primary RP and in SSc [12, 13]. In brief, a drop of fingertip blood for lactic acid determination is obtained in resting conditions (pre-CS-FTL) and after CS (post-CS-FTL). SSc patients show higher pre-CS-FTL than do normal controls and primary RP patients (2.13 ± 1.02, 1.59 ± 0.62, 1.61 ± 0.60 mg/dl, respectively). After the CS, there is a decrease in FTL in normal controls (1.23 ± 0.44 mg/dl) and no expressive modification in patients with primary RP (1.58 ± 0.86 mg/dl). Strikingly, SSc patients present an additional increase in FTL after the CS (3.23 ± 1.66 mg/dl), which emphasizes the deleterious effects of cold exposure on the metabolism at the microcirculation level in these patients [12, 13].

In the present study, we have evaluated for the first time how the acute vasodilator effect of nifedipine in the microvascular dynamics of patients with RP can be assessed by the determination of FTL before and after CS in patients with primary RP and in patients with SSc.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Study design
This was a randomized, placebo-controlled, double-blinded crossover study. After randomization, patients received a single sublingual dose of placebo or nifedipine (10 mg) in identical presentation. CS-FTL tests were performed before and 1 h after sublingual nifedipine or placebo administration. After a 2-week washout period, patients were crossed over to receive the alternative medication and the CS-FTL tests were repeated as previously.

Patients
Twenty patients with primary RP and 20 patients with RP secondary to SSc meeting the American College of Rheumatology preliminary criteria [14] were consecutively selected from the Scleroderma Spectrum Outpatient Clinic at UNIFESP Medical School Hospital. Primary RP was defined according to the criteria proposed by LeRoy and Medsger [1]. All primary RP patients had been followed for at least 2 yrs and no underlying disease could be identified in this period. Exclusion criteria were the existence of active fingertip ulceration, smoking, occupational exposure to cold environment and to vibratory tools, systemic arterial hypertension, diabetes mellitus, heart failure, renal insufficiency, carpal tunnel syndrome, clinical evidence of proximal arterial disease, history of intolerance to nifedipine and prior digital or proximal sympathectomy. Patients stopped oral vasodilators and any other medication for RP 2 weeks prior to the procedure. All individuals filled out an informed consent form approved by the UNIFESP Ethics Committee. Ethical approval was obtained from the local ethics committee for the whole study.

CS-FTL test and therapeutic protocol
The CS-FTL procedure consisted of four phases. In phase I (stabilization), patients rested for 60 min at 24 ± 1°C. Next, in phase II (pre-drug CS-FTL test), patients were asked to choose the finger with the most severe RP for the FTL measurements. If the patient chose no finger, then the procedure would be performed on the fourth finger of the left hand. FTL was determined before (pre-CS-FTL) and after a CS (post-CS-FTL). Briefly, a brisk puncture was performed at the volar surface of the fingertip with an automatic device (Softclix, Boehringer-Mannheim, Germany) and the first blood drop was adsorbed on to a lactate strip (Boehringer-Mannheim, Germany). The strip was processed immediately in a portable spectrophotometer (Accusport, Boehringer-Mannheim, Germany). Next, patients submerged both hands in water at 10°C for 1 min (CS). FTL was again determined 10 min after CS (post-CS-FTL). In phase III (therapeutic intervention), after a 1 h re-warming period at 24 ± 1°C, patients received a 10 mg nifedipine or identical placebo capsule for sublingual administration. In phase IV (post-drug CS-FTL test), the whole procedure performed in phase II was repeated on the same fingertip 1 h after therapeutic intervention, resulting in a second pre- and post-CS-FTL measurement. After the 2-week washout period, the whole protocol was repeated, this time with the alternative medication.

The difference between post- and pre-CS-FTL (designated {Delta}CS-FTL) was expressed as the percent variation relative to pre-CS-FTL and was defined by the following formula:


Formula

Statistical analysis
Differences in pre-CS-FTL, post-CS-FTL and {Delta}CS-FTL before and after treatment in primary RP and SSc groups were analysed by Wilcoxon's test. Differences between groups were analysed by the Mann–Whitney test. Intra-individual comparisons of placebo vs nifedipine were performed due to the crossover design. Statistical significance level was set at 5% (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Patients with primary RP (20 women) were younger (mean age 34 ± 9.2 vs 45.5 ± 12.5 yrs; P = 0.002) and had shorter disease duration (mean disease duration of 7 ± 8.6 vs 9.9 ± 13.3 yrs; P = 0.01) as compared with SSc patients (15 women and 5 men). Seven (35%) patients had diffuse cutaneous SSc and 13 (65%) limited cutaneous SSc.

Before therapeutic intervention, patients with primary RP and SSc behaved differentially in the CS-FTL test as previously demonstrated [12, 13], i.e. FTL decreased significantly after the CS in patients with primary RP ({Delta}CS-FTL = –21.3 ± 13.0 and –18.8 ± 16.6% before placebo and before nifedipine, respectively) and increased significantly in SSc patients ({Delta}CS-FTL = +24.5 ± 21.2 and +15.9 ± 24.7% before placebo and before nifedipine, respectively) (Table 1). There was no significant difference between pre-CS-FTL before placebo vs before nifedipine (primary RP, P = 0.658; SSc, P = 0.244), between post-CS-FTL before placebo vs before nifedipine (primary RP, P = 0.551; SSc, P = 0.491) or between {Delta}CS-FTL before placebo vs before nifedipine (primary RP, P = 0.66; SSc, P = 0.26) in both groups of patients. These observations argue for the reproducibility of the CS-FTL test when performed 2 weeks apart.


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  		TABLE 1. Pre-CS-FTL, post-CS-FTL and {Delta}CS-FTL (mean ± S.D., median) in primary RP and in SSc patients before and after the therapeutic interventions with placebo or nifedipine

 
As expected, placebo had no effect on CS-FTL parameters in both groups of patients (Table 1). Conversely, nifedipine decreased significantly pre-CS-FTL and post-CS-FTL, but not {Delta}CS-FTL, in patients with primary RP. In SSc patients, nifedipine could not alter pre-CS-FTL but was able to reverse the aberrant behaviour of SSc patients after the CS. Before nifedipine, SSc patients presented an increase in FTL after CS and therefore a positive {Delta}CS-FTL, but after the use of nifedipine there was a decrease in FTL after CS and therefore a negative {Delta}CS-FTL. The difference between post-CS-FTL and {Delta}CS-FTL obtained before and after nifedipine in SSc was statistically significant (Table 1). The spectrum of inter-individual variation in pre-CS-FTL, post-CS-FTL and {Delta}CS-FTL before and after the use of nifedipine in patients with RP and SSc is depicted in Fig. 1. Taking advantage of the crossover design, we observed that in primary RP patients, the intra-individual comparison for pre-CS-FTL showed lower figures after nifedipine vs after placebo (P = 0.016), but no statistically significant difference was found for post-CS-FTL and {Delta}CS-FTL (P = 0.111 and P = 0.217, respectively). In contrast, in SSc patients, the intra-individual comparison for {Delta}CS-FTL and for post-CS-FTL, but not for pre-CS-FTL, after nifedipine vs after placebo, showed a statistically significant difference (P < 0.001, P = 0.032 and P = 0.61, respectively).


Figure 1
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  		FIG. 1. Distribution of patients with primary RP and with SSc according to the behaviour of pre-CS-FTL (A, D), post-CS-FTL (B, E) and {Delta}CS-FTL (C, F) before and after the use of nifedipine. Grey lines link individual patients. Adjacent box-plot graphs depict the overall behaviour of the groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we showed that the CS-FTL test could discriminate the effects of placebo and nifedipine on the peripheral perfusion in SSc and in RP patients. In patients with SSc, nifedipine caused a striking improvement in the peripheral perfusion after cold exposure but could not alter significantly the resting peripheral perfusion. In patients with primary RP, nifedipine had a positive effect on the peripheral perfusion in resting conditions and after a CS. Although the mean age and the duration of RP values were slightly different in the SSc and RP patients, this small difference was not felt to represent a problem since we have previously demonstrated that age and gender do not influence significantly CS-FTL test parameters [13].

The CS-FTL test is a reproducible quantitative method that provides a biochemical parameter to evaluate the effects of cold exposure on the microcirculation in patients with primary and secondary RP [12, 13]. The data herein reported confirm our previous findings that FTL is increased in resting conditions in patients with SSc and that it is further increased after a CS. This is in frank opposition to normal subjects and to patients with primary RP in whom FTL decreases after a CS. In addition, we have observed that a single sublingual nifedipine dose was able to improve lactate parameters in primary RP and in SSc patients. In primary RP patients, nifedipine was able to decrease resting and post-CS-FTL. This is expected since the increased sympathetic nervous system activity and the resulting vasoconstriction are the principal factors responsible for the digital vasospasm in primary RP. In SSc patients, nifedipine could not affect the resting FTL, which is not unexpected since calcium channel blockers are not supposed to interfere with structural vessel abnormalities and SSc patients have marked intimal thickening and luminal narrowing of small arteries and arterioles [15–17]. However, we also observed that nifedipine had a striking effect on FTL after a CS in patients with SSc. Under the effect of nifedipine, the paradoxal increase in FTL after CS normally observed in SSc patients was reversed to the normal pattern, i.e. a decrease in FTL after the CS. This observation suggests that calcium channel blockers may still be beneficial in SSc patients by diminishing the intensity of the superimposed cold-induced vasospasm on the already structurally impaired microcirculation. The observation that nifedipine had a striking effect on post-CS-FTL in SSc patients is relevant since these patients are especially vulnerable to the effects of cold exposure. In the long run, it is possible that the regular use of oral calcium channel blockers may lessen the effects of chronic ischaemia in the extremities of these patients.

The low intra-individual variability of the CS-FTL test could be verified by the measures before and after placebo. We have previously observed that in normal subjects, there was no significant difference in CS-FTL determinations obtained in the same individual within a 1 h interval [13]. In the present study, the intra-individual comparisons between FTL measurements before and after two cold stimuli within a 1 h interval under placebo have confirmed the reproducibility of the CS-FTL test. This observation also indicates that the 1 h re-warming period was enough to restore the basal metabolism in primary RP and in SSc patients. In addition, the absence of significant intra-individual difference between FTL measurement before intervention with placebo and nifedipine 2 weeks apart (crossover) argues for the reproducibility of the CS-FTL test when performed on separate occasions. This is an important point since it supports the use of repetitive CS-FTL test as an evaluation parameter in therapeutic protocols. However, this point needs long-lasting longitudinal studies on a larger number of patients.

In conclusion, we have demonstrated that the CS-FTL test was able to detect the acute effect of sublingual nifedipine on the biochemical dimension of the peripheral perfusion and that a single nifedipine dose caused a significant improvement in tissue perfusion in primary RP and in SSc patients. The present results suggest that the CS-FTL test is a relevant tool to monitor therapeutic interventions in patients with primary RP and SSc. Further studies are warranted to evaluate the effects of the chronic use of nifedipine and other peripheral vasodilators on the biochemical peripheral metabolism in primary RP and in SSc.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
Funding: This study was financially supported by The State of São Paulo Research Foundation (FAPESP) and the Brazilian Society of Rheumatology Research Agency.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 27 May 2007; revised version accepted 10 October 2007.
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