A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevulinic acid-n-pentylester.

BACKGROUND: Topical 5-aminolaevulinic acid (ALA) is widely used in photodynamic therapy (PDT) to generate protoporphyrin IX (PpIX) in the skin. However, other prodrugs may be more effective. OBJECTIVES: The pharmacokinetics of ALA- and ALA-n-pentylester-induced PpIX, together with the phototoxicity after PDT, were compared in human skin in vivo, using iontophoresis as a quantitative drug delivery system. METHODS: A series of six increasing doses of equimolar prodrug solutions was iontophoresed into normal skin of the upper inner arms of 20 healthy subjects. The kinetics of PpIX metabolism in skin (n = 4) and the response to light exposure, performed at 4.5 h (n = 6) and 6 h (n = 10) after application, were assessed by skin surface PpIX fluorescence and postirradiation erythema. RESULTS: ALA and ALA-n-pentylester showed a linear correlation between logarithm of dose and PpIX fluorescence (P < 0.005), and logarithm of dose and skin phototoxicity with irradiation at 4.5 h (P < 0.001 and P < 0.005, respectively) and 6 h (P < 0.05 and P < 0.0001, respectively) after iontophoresis. Higher phototoxicity was observed with ALA-n-pentylester than with ALA when sites were irradiated at 6 h, as indicated by the significantly lower theoretical threshold dose for erythema (P < 0.05) and the shift of the PpIX fluorescence/phototoxicity curve towards greater skin erythema at equal PpIX fluorescence levels. Depth of PpIX fluorescence in skin, as determined by fluorescence microscopy, was similar for both prodrugs, but a more homogeneous distribution of PpIX was seen with the more lipophilic ALA-n-pentylester. CONCLUSIONS: The observed greater phototoxicity of ALA-n-pentylester relative to ALA may be attributable to a more favourable PpIX localization in tissue and/or greater intrinsic toxicity.

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo.

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.

Pressure versus heat-induced unfolding of ribonuclease A: the case of hydrophobic interactions within a chain-folding initiation site.

To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.

The role of phenylalanine 31 in maintaining the conformational stability of ribonuclease P2 from Sulfolobus solfataricus under extreme conditions of temperature and pressure.

Ribonuclease P2 from the thermophilic archaebacterium Sulfolobus solfataricus is a small protein (7 kDa) with a known three-dimensional structure. Inspection of the structure and molecular dynamics simulation reveal that three aromatic residues (Phe5, Phe31, and Tyr33) from the hydrophobic core have a strong van der Waals interaction energy. We studied the thermodynamics of the heat, cold, and pressure-induced protein conformational changes of the wild type and of the F31A and F31Y mutants by analyzing the protein UV absorbance in the fourth derivative mode. The wild-type protein was extremely stable under all conditions of temperature and pressure. Heat and cold denaturation of both mutants, as well as denaturation by pressure of the F31A mutant, led to significant blue shifts of the derivative spectrum, indicating increased solvent exposure of Tyr33. For the F31Y mutant, high pressure (400 MPa) protected the protein against thermal denaturation. This study, probing the properties of the hydrophobic aromatic core, complements a thermal unfolding study which probes the overall structural changes [Knapp, S., Karshikoff, A., Berndt, K. D., Christova, P., Atanasov, B., & Ladenstein, R. (1996) J. Mol. Biol. 264,1132-1144]. The differences observed in response to extremes of temperature, pressure, and pH may be rationalized by an unfolding mechanism involving larger parts of the peripheral protein while the integrity of the hydrophobic core is maintained.

Femtosecond transient absorption study of carotenoid to chlorophyll energy transfer in the light-harvesting complex II of photosystem II.

Singlet energy transfer between the carotenoids (Cars) and chlorophylls (Chls) in the light-harvesting complex II (LHC II) from higher plants has been studied using ultrafast transient absorption spectroscopy by exciting the Cars directly in the 475-515 nm wavelength range. LHC II trimers from Arabidopsis thaliana with well-defined Car compositions have been used. From HPLC, the wild type (WT) monomer contains two luteins (Ls), one neoxanthin (N), and a trace of violaxanthin (V) per 12 Chls. The ABA-3 mutant contains 1.4 Ls and 0.6 zeaxanthin (Z) per monomer. Though exploitation of the difference in Car constitution and exciting the WT at 475 and 490 nm, and the ABA-3 mutant at 490 and 515 nm, the different Car contributions to energy transfer have been probed. Evidence for energy transfer mainly from the Car to Chl b is observed in the WT. In the mutant, additional transfer from Car to Chl a correlates with the presence of Z. The results imply predominant energy transfer from the central Ls to Chl b which requires a modification of the currently accepted arrangement of Chl pigments in LHC II.

Monitor lyophilization with mass spectrometer gas analysis.

This paper presents a brief review of the basic issues of lyophilization and the fundamentals of quadrupole mass spectrometry. The emphasis of the paper is on the application of a quadrupole mass spectrometer as a process monitor. The details of sampling and data collection are discussed. Data taken while monitoring lyophilization is presented in conjunction with cycle optimization, end point detection and contaminant detection.

The extent of physician participation in Medicaid: a comparison of physician estimates and aggregated patient records.

This article compares two measures of the extent of physician participation in Medicaid programs. The first, which has been used in most research to date on the subject, is based on physician estimates of the proportion of their patients who are Medicaid patients. The second derives from encounter forms for a sample of visits to the interviewed physicians. The comparison shows that physicians in the sample tended to overestimate by 40 percent the extent of their Medicaid participation. Because the two measures are highly correlated, the analysis of the determinants of Medicaid participation was not affected by the measure used. However, since physicians tended to overstate the proportion of Medicaid patients in their practices, interview data should not be used to measure the amount of physician participation or to calculate elasticities for the effects of policy changes on the extent of participation.

Full and limited medicaid participation among pediatricians.

Participation in Medicaid by pediatricians is an important element in the access of low income children to health care. Factors that influence whether participating pediatricians choose to participate fully in the program or to limit their acceptance of Medicaid patients are identified and analyzed. Data were derived from interviews conducted with 814 pediatricians in 13 states. A multivariate analysis examining physician, practice, service area, and Medicaid policy characteristics indicates that policy factors are most influential in the physician's decision whether to participate fully in medicaid programs. Factors found to foster the willingness of pediatricians to participate fully in state Medicaid programs included more competitive levels of reimbursement, minimal delays in reimbursement, and eligibility and benefit policies that minimize interference with the exercise of medical judgment.

Current distribution and trends in the location pattern of pediatricians, family physicians, and general practitioners between 1976 and 1979.

The literature suggests that pediatricians in the United States are concentrated in the more densely populated regions and states, whereas family physicians and general practitioners are more likely to settle in rural areas. The rapidly increasing supply of all child health physicians had led many to hypothesize that the traditional geographic preferences of pediatricians would expand to include smaller communities. Data for 1976 to 1979 confirm the urban concentration of pediatricians and the more even distribution of family physicians and general practitioners. These data also demonstrate a marked imbalance of pediatricians within county groups, resulting in some areas of shortage even within highly metropolitan communities. Evidence of a trend toward increased dispersion of pediatricians into urban shortage areas is presented, but there is no indication that enough pediatricians will settle in rural areas to meet the needs of children in those small communities.

Nonphysician health care providers in pediatrics.

This paper has identified nurse practitioners, physician's assistants and, potentially, nurse-midwives, as nonphysician health care providers who might have a significant impact on physician manpower requirements in pediatrics in the United States. These providers are capable of providing well child care, as well as care for minor illnesses. Patient acceptance and the quality of care rendered by these providers appear to be high. Physician acceptance is also high, especially when the physician has actually employed a nonphysician provider. State legislation and reimbursement procedures may interfere with the full utilization of nonphysician providers, but when nurse practitioners and physician assistants are employed in pediatric care, they deliver a wide range of services efficiently and economically. There are estimated to be a total of approximately 5,500 nonphysician providers specializing in pediatric care, but other nonphysician providers who may also care for children are discussed. The total number of these providers for 1980 is estimated at 7,232 and for 1990, at 15,512.