#BeautyRecap: June 19th, 2018

Research and Technology

Knowledge removes discomfort
JAMA

Immunotherapy and skin side effects
JAMA

The ethical issue of “cherry picking” patients
JAAD

Articles of interest to dermatologists from the nondermatologic literature
JAAD

Cross-sectional assessment of ultraviolet radiation–related behaviors among young people after a diagnosis of melanoma or basal cell carcinoma
JAAD

Keeping an eye on atopic dermatitis
JAAD

Dermatoses caused by cultural practices
JAAD

2-methoxymethyl-para-phenylenediamine containing hair dye as a less allergenic alternative for para-phenylenediamine allergic individuals
JAAD

Widespread regular sunscreen application deemed not useful in the U.S.A.
British Journal of Dermatology

Deep learning-based, computer-aided classifier developed with a small dataset of clinical images surpasses board-certified dermatologists in skin tumor diagnosis
British Journal of Dermatology

Widespread regular sunscreen application deemed not useful in the U.S.A.: reply from authors
British Journal of Dermatology

Why a randomized melanoma screening trial is not a good idea
British Journal of Dermatology

The global prevalence and correlates of skin bleaching: A meta-analysis and meta-regression analysis
International Journal of Dermatology

Polyamine regulator AMD1 promotes cell migration in epidermal wound healing
Journal of Investigative Dermatology

Dermal fibroblast SLC3A2 deficiency leads to premature aging and loss of epithelial homeostasis
Journal of Investigative Dermatology

The in vitro antimicrobial evaluation of commercially essential oils and their combinations against acne
International Journal of Cosmetic Science

Colour differences in Caucasian and Oriental women’s faces illuminated by white light‐emitting diode sources
International Journal of Cosmetic Science

Human skin‐depigmenting effects of resveratryl triglycolate, a hybrid compound of resveratrol and glycolic acid
International Journal of Cosmetic Science

Validation of an in vitro sun protection factor (SPF) method in blinded ring‐testing
International Journal of Cosmetic Science

The treatment of hyaluronic acid aesthetic interventional induced visual loss (AIIVL): A consensus on practical guidance
Journal of Cosmetic Dermatology

A population ‐based cohort study of atopic eczema among young adult males in Singapore
Journal of Cosmetic Dermatology

Acne treatment with no side effects
New Scientist

Two important novelties in etiopathogenesis and therapy of acne
JEADV

Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates
JEADV

Characterisation of Cutibacterium acnes phylotypes in acne and in vivo exploratory evaluation of Myrtacine®
JEADV

Effectiveness and safety of an overnight patch containing Allium cepa extract and Allantoin for post-dermatologic surgery scars
Aesthetic Plastic Surgery

Protective effect of crocin on ultraviolet B ‑induced dermal fibroblast photoaging
Molecular Medicine Reports

Oxybenzone and solar filters in general: The good and the bad
Actas Dermo-Sifiliograficas

Innovative natural ingredients-based multiple emulsions: The effect on human skin moisture, sebum content, pore size and pigmentation
Molecules

Stabilizing the microbiome skin-gut-brain axis with natural plant botanical ingredients in cosmetics
Cosmetics

Involvement of the nuclear structural proteins in aging-related responses of human skin to the environmental stress
Clinical, Cosmetic and Investigational Dermatology

Skin penetration of Vitamin C (Ascorbic acid): Part II

“Applying 15% Vitamin C for three consecutive days creates a reservoir effect in the skin.”

Firstly, I want to remind you that this study was done on pig skin – not humans. The way that ascorbic acid is stored and metabolized in pig skin may vary from human skin.

Most animals, like pigs, are able to synthesize their own ascorbic acid from glucose, but humans cannot. It’s possible that this data from pigs will be similar to human data, but it’s also very possible that it won’t be. Neither has been proven yet. Presenting an assumption as truth is misleading – but often done in marketing.

I also want to remind you that the way that the ascorbic acid was applied to the skin was not the same way that we apply our skincare. In these experiments, the ascorbic acid solution was applied with a Hill Top Chamber, which occludes the skin, reducing evaporation and theoretically enhancing skin penetration.

For this part of the experiment, Pinnell and his group applied a 15% ascorbic acid solution at pH 3.2 to pig skin for 5 days with a Hill Top Chamber. After the 5th day, application of the ascorbic acid was stopped and ascorbic acid levels in the skin were monitored for an additional 5 days.

After the 3rd day of application of the ascorbic acid serum, the ascorbic acid levels in the skin do appear to reach a peak around 1100 pmol/mg. The deviation around the mean does appear to be reducing with each further day between the 3 subject pigs.

We do need to consider if this theoretical peak amount of ascorbic acid is reached in real-life situations. The living conditions of the pigs in the study were not described, so it’s possible that they were not exposed to natural daylight. It’s understood that UV exposure reduces the amount of ascorbic acid in the skin. UV increases the production of free radicals in the skin, and ascorbic acid is part of the natural antioxidants in the skin which help neutralize these free radicals.

In an experiment using human skin models, it was found that exposure to 16.9 joules/cm² (About 12 minimal erythemal dose equivalent) of UV reduced ascorbic acid levels in the skin model by almost ⅓. This was a higher amount of UV exposure the experimenters expected, they were also unable to detect dehydroascorbic acid in the skin. The study does have some issues which “may be explained by the high levels of ascorbate present in the [tissue] medium…added by the manufacturer to increase collagen synthesis”.

“Vitamin C remains in the skin for 3-4 days and doesn’t wash out”

This marketing claim may be due to some confusion of the term “washout”. In drug experiments a “washout period” refers to the period of time when treatment is stopped, it does not necessarily mean that the skin is washed out.

After applying the 15% ascorbic acid solution to the pig skin, they discontinued application and monitored ascorbic acid levels in the skin. Unfortunately, the methodology in this portion of the experiment isn’t explicitly described. It is unclear, for example, if the pig’s skin was washed each day. The washing procedure is described as “…at the end of the experiment, the formulation was washed vigorously from the skin with water.”

Because most of us use surfactant based cleansers to wash our skin, this data may not be as applicable as the pig’s skin was washed with only water. However, the pig’s skin was removed of stratum corneum before ascorbic acid measurement and the lower layers of skin are likely less affected by the washing and surfactant based-cleanser.

Based on this data, the half-life (the amount of time it takes for the detected ascorbic acid levels to drop by half) was estimated at around 4 days. But as mentioned above, it’s unclear what the living conditions of the pigs were and whether or not they were exposed to sunlight which reduces antioxidant levels in the skin.

Can Vitamin C derivatives increase levels of Vitamin C in skin?

The last portion of the Pinnell experiment looked at whether or not the topical application of Vitamin C derivatives could increase levels of Vitamin C as ascorbic acid in pig skin.

For 24 hours, solutions of dehydroascorbic acid, 10% ascorbyl-6-palmitate, 12% magnesium ascorbyl phosphate, and 15% ascorbic acid were applied to pig skin. Compared to control, only the 15% ascorbic acid solution created a statistically significant increase in ascorbic acid levels in the skin.

For the derivatives, there was no statistically significant difference between the application of the derivative and control (no application of derivatives or Vitamin C) – which implicates that, at least for pig skin, these specific derivatives do not convert to Vitamin C.

For the solutions of dehydroascorbic acid, pig skin levels of ascorbic acid were 7.51 ± 3.34 pmol/mg for 20 mM dehydroascorbic acid and 8.70 ± 2.13 pmol/mg for 1 M dehydroascorbic acid. Where no dehydroascorbic acid was applied levels of ascorbic acid were 9.24 ± 3.55 pmol/mg.

In conclusion…

It surprises me how influential this one study on ascorbic acid applied to pig skin has become in terms of marketing language for brands.

Even later studies with Dr. Pinnell as an author leave out that the data are collected from pig skin, “Following topical application, once the skin is saturated with L-ascorbic acid, it remains with a half-life of about 4 d (Pinnell et al, 2001).”

While this experiment is some of the best data we have in terms of ascorbic acid penetration based on formulation, the key point to remember is that human skin cannot be assumed to behave the same as pig skin.

So if you see a claim similar to “15% Vitamin C at pH 3.5 is the most effective concentration”, please imagine me beside you whispering “…for pigs”.

Edit: An error was made in the original version published, pigs can synthesize Vitamin C from glucose, but humans can not. Guinea pigs also cannot synthesize their own Vitamin C.

Edit: An error was made in the original version published, pmmol was corrected to pmol.

Source: Pinnell, S. R., Yang, H. , Omar, M. , Riviere, N. M., DeBuys, H. V., Walker, L. C., Wang, Y. and Levine, M. (2001), Topical L‐Ascorbic Acid: Percutaneous Absorption Studies. Dermatologic Surgery, 27: 137-142. DOI: 10.1046/j.1524-4725.2001.00264.x

Podda, M., Traber, M.G., Weber, C., Yan, L., Packer, L. (1998), UV-Irradiation Depletes Antioxidants and Causes Oxidative Damage in a Model of Human Skin, Free Radical Biology and Medicine, 24: 55-65. DOI: 10.1016/S0891-5849(97)00142-1

#BeautyRecap: June 12th, 2018

Research and Technology

Tretinoin (0.05% cream vs. 5% peel) for photoaging and field cancerization of the forearms: randomized, evaluator‐blinded, clinical trial
JEADV

pH in nature, humans and skin
Journal of Dermatology

Platelet rich plasma for photodamaged skin: A pilot study
Journal of Cosmetic Dermatology

Cutaneous acceptability of a moisturizing cream in subjects with sensitive skin
Journal of Cosmetic Dermatology

Ionizing radiation, but not ultraviolet radiation, induces mitotic catastrophe in mouse epidermal keratinocytes with aberrant cell cycle checkpoints
Experimental Dermatology

Mechanical spectroscopy and imaging of skin components in vivo: Assignment of the observed moduli
Skin Research & Technology

Validation of GPSkin Barrier for assessing epidermal permeability barrier function and stratum corneum hydration in humans
Skin Research & Technology

Photoallergic contact dermatitis to sunscreens containing oxybenzone in La Plata, Argentina
Actas Dermo-Sifiliograficas

Clinical and dermoscopic evaluation of combined (salicylic acid 20% and azelaic acid 20%) versus trichloroacetic acid 25% chemical peel in acne: a RCT
Journal of Dermatological Treatment

Schisandra chinensis protects the skin from global pollution by inflammatory and redox balance pathway modulations: An in vitro study
Cosmetics

Is maintenance treatment in adult acne important? Benefits from maintenance therapy with adapalene, and low doses of alpha and beta hydroxy acids
Journal of Dermatological Treatment

Platelet rich plasma with microneedling and trichloroacetic acid peel for treatment of striae distensae
JAAD

Single-center, double-blind, randomized, placebo-controlled, study of the efficacy and safety of a cream formulation for improving facial wrinkles and skin quality
Journal of Drugs in Dermatology

Who is accountable when patients do not achieve successful treatment for their acne?
Journal of Drugs in Dermatology

The efficacy and safety of azelaic acid 15% foam in the treatment of facial acne vulgaris
Journal of Drugs in Dermatology

Clinical experience with once-daily Dapsone gel, 7.5% monotherapy in patients with acne vulgaris
Journal of Drugs in Dermatology

The effect of an anti-inflammatory botanical cleanser/night mask combination on facial redness reduction
Journal of Drugs in Dermatology

Antiaging effects of a novel facial serum containing L-ascorbic acid, proteoglycans, and proteoglycan-stimulating tripeptide: Ex vivo skin explant studies and in vivo clinical studies in women
Clinical, Cosmetic and Investigational Dermatology

Chain dynamics of human dermis by Thermostimulated currents: A tool for new markers of aging
Skin Research & Technology

Effects of atopic dermatitis and gender on sebum lipid mediator and fatty acid profiles
PLEFA

Risk of sun‐induced skin cancers in patients with alopecia areata, alopecia totalis and alopecia universalis
JEADV

The effects of season and weather on healthcare utilization among patients with atopic dermatitis
JEADV

Body image, personality traits, and quality of life in botulinum toxin a and dermal filler patients
Aesthetic Plastic Surgery

From mice to men: An evolutionary conserved breakdown of the epidermal calcium gradient and its impact on the cornified envelope
Cosmetics

Mild hyperbaric oxygen activates the proliferation of epidermal basal cells in aged mice
The Journal of Dermatology

Indian irrational skin creams and steroid‐modified dermatophytosis – an unholy nexus and alarming situation
JEADV

#BeautyRecap: June 5th, 2018

Research and Technology

Determining the clinical relevance of positive patch testing to gold in evaluation of contact dermatitis
The Journal of Allergy and Clinical Immunology: In Practice

Do sunscreen prevent recurrent herpes labialis in summer?
Journal of Dermatological Treatment

Adolescents’ perspectives on atopic dermatitis treatment: Experiences, preferences, and beliefs
JAMA Dermatology

Prevalence of skin cancer examination among indoor tanning bed users
JAMA Dermatology

The spectrum and sequelae of acne in Black South Africans seen in tertiary institutions
Skin Appendage Disorders

Antiaging effects of a novel facial serum containing L-ascorbic acid, proteoglycans, and proteoglycan-stimulating tripeptide: ex vivo skin explant studies and in vivo clinical studies in women
Clinical, Cosmetic and Investigational Dermatology

Differential roles of RAD18 and CHK2 in genome maintenance and skin carcinogenesis following UV exposure
Journal of Investigative Dermatology

Assessment of the effect of 3% Diclofenac Sodium on photodamaged skin by means of reflectance confocal microscopy
Acta Dermato-Venereologica

The beneficial effect of Korean Red Ginseng extract on atopic dermatitis patients: An 8 weeks open, noncomparative clinical study.

Deeper wrinkle formation and less melanin production in aged Korean women with B blood type
Annals of Dermatology

Airborne allergic contact dermatitis caused by neem oil
Actas Dermo-Sifiliograficas

Investigating the therapeutic potential of a probiotic in a clinical population with chronic hand dermatitis
Clinical, Cosmetic and Investigational Dermatology

Vitamin D levels in acne vulgaris patients treated with oral isotretinoin
Journal of Cosmetic Dermatology

#BeautyRecap: May 29th, 2018

Products and Reviews

A look at the Kat Von D Lip Liner Vault
Elle.com

Manny MUA reveals his new beauty line Lunar Beauty
Glamour.com

Spectrum launches brush collaboration with Disney’s “The Little Mermaid”
Allure.com

Boscia launches a cactus water moisturizer
Allure.com

Beautyblender launches in new blue shade
TeenVogue.com

15 women of colour-owned beauty brands
Elle.com

Nikita Dragun becomes the face of new Morphe lipstick collection
Elle.com

Peach and Lily launches its own skincare range
Allure.com

Saks Fifth Avenue reveals new New York beauty floor
VanityFair.com

Skincare and Beauty

Sephora launching makeup classes for the transgender community
Elle.com

Restylane Lyft approved by the US FDA for hand rejuvenation
Allure.com

US FDA issues warning about ‘sunscreen pills’
Glamour.com

“Does natural skincare work?” by Cotton Codinha for Elle
Elle.com

US Supreme Court will not hear hair locs workplace lawsuit
TeenVogue.com

Dermatologists recommend their favourite sunburn remedies
Vogue.com

Industry

Debra Perelman to be named first female CEO of Revlon
Allure.com

Rosie Huntington-Whiteley launching her own beauty brand Rose Inc.
Allure.com

Target launches online ‘Beauty Studio’
Glamour.com

Research and Technology

Rethinking the prescription of biotin for dermatologic conditions
Dermatologic Therapy

Beneficial effects of antioxidant furfuryl palmitate in non-pharmacologic treatments (Prescription emollient devices, PEDs) For atopic dermatitis and related skin disorders
Dermatology and Therapy

Hybrid diffuse reflectance spectroscopy: Non-erythemal in vivo testing of sun protection factor
Skin Pharmacology and Physiology

The impact of acne and facial post-inflammatory hyperpigmentation on quality of life and self-esteem of newly admitted Nigerian undergraduates
Clinical, Cosmetic and Investigational Dermatology

Japanese Dermatological Association guidelines: Guidelines for the treatment of acne vulgaris
The Journal of Dermatology

Towards a reliable, non‐invasive melanin assessment for pigmented skin
Skin Research and Technology

Prediction of skin anti‐aging clinical benefits of an association of ingredients from marine and maritime origins: Ex vivo evaluation using a label‐free quantitative proteomic and customized data processing approach
Journal of Cosmetic Dermatology

A single center, prospective, randomized, sham-controlled, double-blinded, split-face trial using microinjections of transparent hyaluronic acid gel for cheek rejuvenation
Dermatologic Surgery

Commentary on a single center, prospective, randomized, sham-controlled, double-blinded, split-face trial using microinjections of transparent hyaluronic acid gel for cheek rejuvenation
Dermatologic Surgery

Importance of dermal absorption of polycyclic aromatic hydrocarbons derived from barbecue fumes
Environmental Science and Technology

Skin penetration of Vitamin C (Ascorbic acid): Part I

Today I wanted to look at a research paper primarily led by Dr. Sheldon R. Pinnell. He is one of the founders of Skinceuticals and contributed much of the early research on the use of Vitamin C as ascorbic acid on skin. He and his group also discovered the synergistic effect of Vitamin C, Vitamin E, and Ferulic acid – which is commonly used in many products on the market today.

The data from this paper is often quoted in marketing material for Vitamin C serums, but one extremely important piece of information is often left out – the data was collected from pigs, white Yorkshire pigs to be exact.

Many people also have ethical concerns when it comes to the use of animals in cosmetic research. Synthetic and lab grown human skin equivalents are being researched and tested which will one day replace the use of animal as well as human testing in cosmetics.

It should be clear that human skin and pig skin are not the same, but they do have similar properties which is why it is often used in experiments. However, one should never assume that data from a pig can be assumed to be the same for a human. The movement and deposition of chemicals often differs between human and pig skin.

From my searches, I haven’t been able to find similar research performed on humans. This paper in particular has led to some of the often quoted “rules” about ascorbic acid.

“Ascorbic acid must have a pH below 3.5 for effective penetration.”

Pinnell and his group tested a 15% ascorbic acid solution adjusted to different pHs ranging from 2 to 5. The 15% ascorbic acid solutions also contained 2% zinc sulfate, 0.5% bioflavonoids, 1% hyaluronic acid, and 0.1% citrate.

While the control situation wasn’t described it’s likely either the vehicle (product without the ascorbic acid) or a water solution was applied to the skin. The control measurement shows that there is some inherent levels of ascorbic acid already present in the skin from the diet.

The test solutions were applied to the pig skin using a Hill Top Chamber. A Hill Top Chamber is a small and round disk which is placed on the surface of the skin, the product is placed in the chamber or a piece of fabric is soaked in the testing material, and the entire chamber is then sealed. This reduces loss of product from evaporation and is a common method of performing occlusive test patches.

The ascorbic acid solutions at pH 2.5, 3.0, 3.5, 4.0, and 5.0 were performed on three pigs, however the control, pH 2, and 4.5 were only performed on two pigs.

The Hill Top Chamber was soaked with 0.2 mL of the ascorbic acid solution then sealed for 24 hours. After this period of occlusion, the skin washed then stripped of the stratum corneum and then small pieces of the skin was removed and tested for ascorbic acid content.

As you can see from the data, the amount of ascorbic acid found in the skin was much higher in ascorbic acid solutions at pH 3.5, 3.0, 2.5, and 2.0. The researchers theorize that it is due to the pKa of ascorbic acid which is 4.2. When the pH of a solution containing ascorbic acid is lower than its pKa more of the ascorbic acid will be protonated. Protonated ascorbic acid is neutrally charged which may allow it to enter the skin more easily.

It’s important to notice the error bars on the amount of ascorbic acid absorbed at pH 2.0. There is considerable deviation from the mean in the results even though it was only tested on 2 subjects. More test subjects would provide a clearer idea of how much ascorbic acid would penetrate at pH 2 on an average population of pigs.

Statistical differences also weren’t calculated between the data points, for example it’s difficult to tell from the way that the data is presented if there is a change in ascorbic acid content between the control, pH 4.0, 4.5, and 5.0 – even if they look different on the graph. Likewise, it’s difficult to tell if there is an increase in ascorbic acid penetration between pH 3.0 and pH 2.5 – despite the trend with pH 2.0 pushing you towards that inference. It’s likely that there is a statistically significant difference between absorption between pH 3.5 and 3.0, but a larger study would provide us  with more confident answers.

So based on this data, many further studies and brands have assumed that a pH below 3.5 results in considerable more skin penetration of ascorbic acid on humans – despite these results being performed on pigs, and relative low strength of the study. If the reason why ascorbic acid is more easily absorbed into the skin is due to the pKa then this would likely hold true for humans as well.

This assumption is often presented as fact, which is misleading. It also doesn’t take into account other factors present in a cosmetic product, such as penetration enhancers. Encapsulation, surfactants, and solvents could increase (or decrease) the amount of ascorbic acid absorbed into the skin regardless of the product’s pH.

In this experiment, the stratum corneum was removed before measurements of ascorbic acid to test for deep penetration of ascorbic acid. It’s possible that some of the benefits conferred by topical application of ascorbic acid aren’t facilitated by deep penetration, the antioxidant and photoprotective effect of ascorbic acid may still occur when it is present in or on the stratum corneum. Other benefits like reduction of hyperpigmentation and an increase in collagen production are likely dependent on penetration past the stratum corneum.

Unfortunately I haven’t been able to find further studies on humans or otherwise to provide answers to these questions.

“Ascorbic acid serums must be at least 10% to be effective”

After the first experiment of testing 15% ascorbic acid with different pHs, Pinnell and his group tested how concentration of ascorbic acid affects skin penetration. This time they tested 7 ascorbic acid solutions with varying concentrations all at pH 3.2. The concentrations of the rest of the formulation are assumed to be the same as the previous experiment.

The ascorbic acid solutions were applied in the same manner, with a Hill Top Chamber for 24 hours, followed by washing, stripping, and then assessment.

The maximum amount of ascorbic acid penetration was seen when 20% ascorbic acid at pH 3.2 was used.

All concentrations were tested on 3 pigs, and there is quite a bit of deviation from mean between absorption among the 3 pigs tested. This makes it difficult to assess the true difference in absorption between a 10% and 15% ascorbic acid, and a 15% and 20% ascorbic acid.

Absorption also seemed to peak at 20%, the 25% ascorbic acid solution penetrated less than the 20%, and the 30% even less so. The researchers did not explore or hypothesize on why this occured, and I’ve been unable to find an answer in any later research as well.

While 20% ascorbic acid certainly led to the greatest increase in levels of ascorbic acid, the 5% solution still increased ascorbic acid levels in the pig skin by about 6 fold.

It’s very important to remember that the way that this experiment was performed does not mimic the way that ascorbic acid solutions are often applied to the skin. With the Hill Top Chamber, the solvent’s (in this case water) evaporation is reduced – whereas when we apply it to the skin the solvent evaporates. What this means is that the kinetics of ascorbic acid penetration into the skin may not be the same.

For example, if half of the solvent of a 10% ascorbic acid solution evaporates, it is equivalent to a 20% ascorbic acid solution – the total amount of ascorbic acid by mass is the same, but the concentration has changed. This may mean that we could see a different maximum absorption by concentration in an experiment where the solvent was allowed to evaporate the way that it is often applied.

Human clinical trials with “low” ascorbic acid concentrations, 3% ascorbic acid cream and a 5% ascorbic acid cream, were able to show statistically significant improvements on measurements of photodamage and photoageing in their study groups.

Another thing many people hold on to is the concept that their products must be working at “maximum efficiency”, unfortunately this is unrealistic and there’s going to be variations in the amount of ascorbic acid that penetrates your skin with each application – even the amount that you apply to your skin will vary each time. This is why good cosmetic studies are performed over a longer period of time.

For example, if we look at the 20% concentration, the pig skin concentration of ascorbic acid increased to about 1100 pmol of ascorbic acid per mg of pig skin, which is about 0.19 μg ascorbic acid per mg of pig skin. 1.0 mg of a 20% ascorbic acid (w/w) contains about 1135589.37 pmol of ascorbic acid, if that helps give you a sense of the “efficiency”. In these experiments, 200 μL or 0.2 mL solution was used in total for each application, which contains about 227117874.1767 pmol of ascorbic acid if we assume density of the solution (w/w) is 1.

Higher concentrations of ascorbic acid may lead to more irritation (measured by skin redness or erythema), but I haven’t found any studies that looked at this specifically.

Continued in Skin penetration of Ascorbic Acid: Part II

Source: Pinnell, S. R., Yang, H. , Omar, M. , Riviere, N. M., DeBuys, H. V., Walker, L. C., Wang, Y. and Levine, M. (2001), Topical L‐Ascorbic Acid: Percutaneous Absorption Studies. Dermatologic Surgery, 27: 137-142. DOI: 10.1046/j.1524-4725.2001.00264.x