HbA1c determination from HemaSpot™ blood collection devices: comparison of home prepared dried blood spots with standard venous blood analysis.

AIM: To assess the clinical performance and patient acceptance of HemaSpot™ blood collection devices as an alternative blood collection method. METHODS: Adult men and women with any type of diabetes, routinely carrying out self-monitoring of blood glucose were recruited (n = 128). Participants provided a venous blood sample and prepared two HemaSpot dried blood spots, one at clinics and one at home. HbA1c analysis was by Tosoh G8 high-performance liquid chromatography. Participants also completed a questionnaire. RESULTS: Strong linear relationships been HbA1c levels in dried blood spots and venous blood were observed and a linear model was fitted to the data. Time between dried blood spot preparation and testing did not impact the model. Participants were accepting of the approach: 69.2% would use this system if available and 60.7% would be more likely to use this system than going to their general practitioner. CONCLUSIONS: The combination of a robust desiccating dried blood spot device, home sample preparation and return by post produces HbA1c data that support the use of a time-independent linear calibration of dried blood spot to venous blood HbA1c . A robust remote sample collection service would be valuable to people living with diabetes in urban areas who are working or house-bound as well as those living in remote or rural locations.

Mechanism of Linolenic Acid-induced Inhibition of Photosynthetic Electron Transport.

The effect of linolenic acid on photosynthetic electron transport reactions in chloroplasts has been localized at a site on the donor side of photosystem I and at two functionally distinct sites in photosystem II.In photosystem I, an increase in the electron transport rate occurs in the presence of 10 to 100 micromolar linolenic acid, followed by a decline in rate from 100 to 200 micromolar linolenic acid. The increase may result from an alteration of membrane structure that allows greater reactivity of the artificial donors 2,6-dichlorophenolindophenol (DPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine with plastocyanin. The decrease is due to loss of plastocyanin from the membrane since addition of purified plastocyanin to treated and washed chloroplasts leads to the reestablishment of photosystem I rates.In photosystem II, a reversible site and an irreversible site of inhibition have been located. At the irreversible site, there is a time-dependent loss of the loosely bound pool of Mn implicated in the water-splitting mechanism. At the reversible site, the photochemical charge separation is rapidly inhibited as evidenced by the high initial fluorescence yield upon illumination and the inhibition of artificial donor reactions in NH(2)OH-washed chloroplasts. When chloroplasts are washed after treatment with linolenic acid, the fluorescence returns to its original low value and there is a resumption of artificial donor activity from diphenylcarbazide --> DPIP. This reversible inhibition of the photoact is a unique characteristic of linolenic acid and suggests evidence for a new mode of inhibition of photosystem II.

Proton evolution from photosystem II. Stoichiometry and mechanistic considerations.

1. In a sequence of flashes given to dark-adapted chloroplasts, the flash yield of proton release oscillates with a period of 4, which is similar but not identical to the oscillation of the O2 flash yield. 2. Using the proton release associated with ferricyanide reduction as a calibration, we computed that two protons are released in the terminal O2-liberating reaction; the other two protons are released in precursor conversion steps. 3. Analysis of the effect of preflashes on the oscillation pattern showed that the S1 leads to S2 transition releases no proton, the S0 leads to S1 transition somewhat less than one (0.75), and the S2 leads to S3 transition somewhat more than one (1.25). 4. The precision of the data was sufficient to exclude the possibility that in the four-step water oxidation, proton release follows a simple 1, 0, 1, 2 pattern. A possible model to interpret the observed flash yield patterns is discussed.

Proton translocation in chloroplasts and its relationship to electron transport between the photosystems.

Using dark adapted isolated spinach chloroplasts and sequences of brief saturating flashes the correlation of the uptake and release of protons with electron transport from Photosystem II to Photosystem I were studied. The following observations and conclusions are reported: (1) Flash-induced proton uptake shows a weak, damped binary oscillation, with maxima occurring after the 2nd, 4th, etc. flashes. The damping factor is comparable to that observed in the O2 flash yield oscillation and therefore explained by misses in Photosystem II. (2) On the average and after a steady state is reached, each flash (i.e. each reduction of Q) induces the uptake of 2H+ from outside the chloroplasts. (3) Flash induced proton release inside the chloroplast membrane shows a strong damped binary oscillation with maximum release occurring also after the 2nd, 4th, etc. flashes. (4) This phenomenon is correlated with the earlier reported binary oscillations of electron transport [2] and shows that both electrons and protons are transported in pairs between the photosystems. (5) In two sequential flashes 4H+ from the outside of the thylakoid and 2e- from water are accumulated at a binding site B. Subsequently, the two electrons are transferred to non-protonated acceptors in Photosystem I (probably plastocyanin and cytochrome f) and the 4H+ are released inside the thylakoid. (6) It is concluded that a primary proton transporting site and/or energy conserving step located between the photosystems is being observed.

Determination of H+/e- ratios in chloroplasts with flashing light.

Using a rapid pH electrode, measurements were made of the flash-induced proton transport in isolated spinach chloroplasts. To calibrate the system, we assumed that in the presence of ferricyanide and in steady-state flashing light, each flash liberates from water one proton per reaction chain. We concluded that with both ferricyanide and methylviologen as acceptors two protons per electron are translocated by the electron transport chain connecting Photosystem II and I. With methyl viologen but not with ferricyanide as an acceptor, two additional protons per electron are taken up due to Photosystem I activity. One of these latter protons is translocated to the inside of the thylakoid while the other is taken up in H2O2 formation. Assuming that the proton released during water splitting remains inside the thylakoid, we compute H+/e- ratios of 3 and 4 for ferricyanide and methylviologen, respectively. In continuous light of low intensity, we obtained the same H+/e- ratios. However, with higher intensities where electron transport becomes rate limited by the internal pH, the H+/e- ratio approached 2 as a limit for both acceptors. A working model is presented which includes two sites of proton translocation, one between the photoacts, the other connected to Photosystem I, each of which translocates two protons per electron. Each site presents a approximately 30 ms diffusion barrier to proton passage which can be lowered by uncouplers to 6-10 ms.

The isolation and characterization of a photochemically active complex from Chloropseudomonas ethylica.

A photochemically active subcellular fraction has been isolated from the green bacterium Chloropseudomonas ethylica. It is enriched in bacteriochlorophyll a and contains only a small amount of Chlorobium chlorophyll. Both a reaction-center bleaching (P840) and a cytochrome oxidation (C553) are observed to occur in the fraction. Particles that are contained in the fraction are larger than 1.5 x 10(6) daltons and exhibit a range of sizes consistent with some form of polymeric association of similar subunits. Around 80 bacteriochlorophyll a molecules are present per P840. In an intact photosynthetic apparatus there are about 1000-1500 total chlorophylls (including Chlorobium chlorophyll) per reaction center. The redox potentials of the reaction center P840 and C553 were found to be 0.24 and 0.17 V, respectively, in one-electron reactions. The potentials of these components are around 0.2 V lower than comparable components found in other photosynthetic organisms.