Steen Brøndsted Nielsen

Microalbuminuria and Potential Confounders: A review and some observations on variability of urinary albumin excretion

U rinary albumin excretion rate (UAER) is the main parameter used in diabetic patients for the clinical evaluation of early renal disease (1-3); likewise, it is a key to other complications (3-5). The parameter is not only related to diagnosis, but it is also important to early intervention, e.g., optimized diabetes care (6), and more specifically, to early antihypertensive treatment (6-10). Several review articles summarize trials documenting that treatment with angiotensin-converting enzyme (ACE) inhibitors (and other antihypertensive agents) in microalbuminuric insulindependent diabetes mellitus (IDDM) and relatively young non-insulin-dependent diabetes mellitus (NIDDM) patients decreases albuminuria and is likely to postpone the decline in glomerular nitration rate (7-9). Optimal diabetic control may be difficult to achieve in some patients with microalbuminuria (11), and the trials in the microalbuminuric patients with this treatment modality do not provide totally coherent results (11-13). However, new data on the microalbuminuric patients in the Diabetes Control and Complications Trial (DCCT) documented a positive effect of intensified treatment (12), but good metabolic control may be problematic in some of these patients, who generally have higher HbAlc values compared with matched normoalbuminuric patients (14). With respect to primary prevention of mi-

The combination of an electrospray ion source and an electrostatic storage ring for lifetime and spectroscopy experiments on biomolecules

An electrospray ion source has been coupled to an accelerator that injects ions into an electrostatic heavy-ion storage ring. Since the dc ion current produced by electrospray ionization is low (∼106 ions/s), ions are accumulated in a cylindrical ion trap filled with a helium buffer gas. The ions are collisionally damped in the buffer gas and confined to the central trap region by a rf field. Extraction from the trap occurs within a few microseconds and after acceleration through 22 kV, the ions of interest are selected by a magnet according to their mass to charge ratio. The ion bunch is subsequently injected into the ring. Both positive and negative ions have been stored, with masses ranging over 3 orders of magnitude (∼102–104 Da). From a pickup signal in the ring, the number of ions in a bunch is estimated to be of the order of 103–104 when the accumulation time is 0.1 s. Our first measurements show that we can store a sufficient number of ions to study the decay of metastable ions and to determine re...

Hidden Histidine Radical Rearrangements upon Electron Transfer to Gas-Phase Peptide Ions. Experimental Evidence and Theoretical Analysis

Protonated peptides containing histidine or arginine residues and a free carboxyl group (His-Ala-Ile, His-Ala-Leu, Ala-His-Leu, Ala-Ala-His-Ala-Leu, His-Ala-Ala-Ala-Leu, and Arg-Ala-Ile) form stable anions upon collisional double electron transfer from Cs atoms at 50 keV kinetic energies. This unusual behavior is explained by hidden rearrangements occurring in peptide radical intermediates formed by transfer of the first electron. The rearrangements occur on a approximately 120 ns time scale determined by the radical flight time. Analysis of the conformational space for (His-Ala-Ile + H)(+) precursor cations identified two major conformer groups, 1a(+)-1m(+) and 5a(+)-5h(+) , that differed in their H-bonding patterns and were calculated to collectively account for 39% and 60%, respectively, of the gas-phase ions. One-electron reduction in 1a(+) and 5a(+) triggers exothermic hydrogen atom migration from the terminal COOH group onto the His imidazole ring, forming imidazoline radical intermediates. The intermediate from 5a is characterized by its charge and spin distribution as a novel cation radical-COO(-) salt bridge. The intermediate from 1a undergoes spontaneous isomerization by imidazoline N-H migration, re-forming the COOH group and accomplishing exothermic isomerization of the initial (3H)-imidazole radical to a (2H)-imidazole radical. An analogous unimolecular isomerization in simple imidazole and histidine radicals requires activation energies of 150 kJ mol(-1), and its occurrence in 1a and 5a is due to the promoting effect of the proximate COOH group. The rearrangement is substantially reduced in Ala-Leu-His due to an unfavorable spatial orientation of the imidazole and COOH groups and precluded in the absence of a free carboxyl group in His-Ala-Leu amide. In contrast to His-Ala-Ile and Arg-Ala-Ile, protonated Lys-Ala-Ile does not produce stable anions upon double electron transfer. The radical trapping properties of histidine residues are discussed.

Systolic Blood Pressure Relates to the Rate of Decline of Glomerular Filtration Rate in Type II Diabetes

OBJECTIVE To relate deterioration in kidney function to some potential cardiovascular risk factors in type II diabetic patients. RESEARCH DESIGN AND METHODS Twenty-four normoalbuminuric and 13 microalbuminuric patients completed a 3.4-yr prospective observational study. Glomerular nitration rate, urinary albumin excretion rate, blood pressure, glycemic control, and lipids were measured on entry and at the end of the study. Of the patients, 19 normoalbuminuric and 8 microalbuminuric (73%) patients had no history of antihypertensive treatment. RESULTS The glomerular filtration rate was significantly reduced during the follow-up period (−1.34 ± 0.54 ml · min−1 · 1.73 m−2 [mean ± SE], P < 0.02). The rate of decline varied considerably in normoalbuminuric and microalbuminuric patients (from −13.5 to 4.3 and from −7.0 to 4.2 ml · min−1 · 1.73 m−2 per year, respectively) but was on average not accelerated in normoalbuminuric or microalbuminuric patients (−1.3 ± 0.7 and −1.5 ± 0.8 ml · min−1 · 1.73 m−2 per year, respectively). Significant correlations were observed between the glomerular filtration rate fall rate and initial systolic blood pressure (r = −0.47, P < 0.01; patients without antihypertensive treatment: r = −0.42, P = 0.03) but not diastolic blood pressure. In a stepwise multiple linear regression analysis, baseline systolic blood pressure significantly determined the fall rate of the glomerular filtration rate (regression coefficient = −0.050, SE = 0.018, P = 0.011; patients without antihypertensive treatment: regression coefficient = −0.047, SE = 0.021, P = 0.030). CONCLUSIONS In these type II diabetic patients neither normoalbuminuria nor microalbuminuria are at an average associated with an accelerated decline in kidney function. Still, systolic blood pressure is a determining factor for the rate of decline in the glomerular filtration rate. A longer follow-up time with consecutive glomerular filtration rate measurements are needed to determine the long-term implications of normoalbuminuria and microalbuminuria on kidney function in type II diabetic patients.

Monitoring Diabetic Nephropathy: Glomerular Filtration Rate and Abnormal Albuminuria in Diabetic Renal Disease—Reproducibility, Progression, and Efficacy of Antihypertensive Intervention

The principal end point in the evaluation of treatment in incipient and overt diabetic nephropathy is rate of decline in glomerular filtration rate (GFR). Therefore, information on reproducibility of GFR measurements is essential in the planning and evaluation of clinical trials. We studied reproducibility of GFR measurements in insulin-dependent and non-insulin-dependent diabetes mellitus patients using, respectively, a constant-infusion technique with urine collection and labeled iothalamate as a tracer marker and a single-shot procedure using Cr-EDTA, measuring the GFR from the decline in plasma level after bolus injection. The coefficient of variance in the insulin-dependent patients was from 7.5% to 8.8% with repeated measurements. In longitudinal studies with several measurements the mean coefficient of variances varied between 7.4% and 3.4%. In the non-insulin-dependent patients the coefficient of variances between two tests were 7.0% and 5.3% for normoalbuminuric and microalbuminuric patients, respectively. In cross-sectional studies as well as in longitudinal studies, it has been consistently shown that GFR is well preserved and at a supranormal level in patients with normoalbuminuria and microalbuminuria. A decline in GFR appears to start around the transition from microalbuminuria to overt diabetic renal disease, although more detailed studies are needed to support this finding. With regard to intervention trials, several studies document that microalbuminuria can be reduced by effective antihypertensive treatment, particularly with angiotensin-converting enzyme inhibitors, also in patients with normal or close to normal blood pressure. Preliminary results from long-term studies suggest that reduction in microalbuminuria in these patients is associated with preservation of GFR and, thus, apparently renoprotection. In patients with overt renal disease, it has been consistently shown that antihypertensive treatment reduces albuminuria as well as the rate of decline in GFR. This is also observed with combined treatment regimens, for instance beta blockers or angiotensin-converting enzyme inhibitors combined with diuretics, or the three types of drugs in combination.

Dynamics of Carbon Monoxide Binding to Cystathionine β-Synthase

Cystathionine β-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 μm-1. Stopped flow measurements reveal slow CO association (0.0166 s-1) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s-1, is essentially unchanged between pH 7.6 and 10.5, indicating that the pKa of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.

Absorption spectrum of the firefly luciferin anion isolated in vacuo.

The excited-state physics of the firefly luciferin anion depends on its chemical environment, and it is therefore important to establish the intrinsic behavior of the bare ion. Here we report electronic absorption spectra of the anion isolated in vacuo obtained at an electrostatic ion storage ring and an accelerator mass spectrometer where ionic dissociation is monitored on a long time scale (from 33 μs and up to 3 ms) and on a short time scale (0-3 μs), respectively. In the ring experiment the yield of all neutrals (mainly CO(2)) as a function of wavelength was measured whereas in the single pass experiment, the abundance of daughter ions formed after loss of CO(2) was recorded to provide action spectra. We find maxima at 535 and 265 nm, and that the band shape is largely determined by the sampling time interval, which is due to the kinetics of the dissociation process. Calculations at the TD-B3LYP/TZVPP++ level predict maximum absorption at 533 and 275 nm for the carboxylate isomer in excellent agreement with the experimental findings. The phenolate isomer lies higher in energy by 0.22 eV, and also its absorption maximum is calculated to be at 463 nm, which is far away from the experimental value. Our data serve to benchmark future theoretical models for bioluminescence from fireflies.

On the Influence of Water on the Electronic Structure of Firefly Oxyluciferin Anions from Absorption Spectroscopy of Bare and Monohydrated Ions in Vacuo

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

Vacuum-ultraviolet circular dichroism spectroscopy of DNA: a valuable tool to elucidate topology and electronic coupling in DNA

Circular dichroism (CD) is a powerful technique to obtain information on electronic transitions and has been used extensively for studies on DNA. Most experiments are done in the UV region but new information is often revealed from extending the wavelength region down into the vacuum ultraviolet (VUV) region. Such experiments are most easily carried out with synchrotron radiation (SR) light sources that provide large photon fluxes. Here we provide a summary of the SRCD data taken on different DNA strands with emphasis on results from our own laboratory within the last five years.(1-3) Signal intensities in the VUV are often significantly larger than those in the UV, and the electronic coupling between bases may increase with excitation energy. CD spectroscopy is particularly useful for investigating the extent of electronic coupling within a strand, i.e., the degree of delocalisation of the excited-state electronic wavefunction. The spatial extent of the wavefunction may be limited to just one base or it extends over two or more bases in a stack or between bases on different strands.(4,5) The actual character of the electronically excited state is linked to base composition and sequence as well as DNA folding motif (A-, B-, Z-DNA, triplexes, quadruplexes, etc.). The latter depends on experimental conditions such as solution acidity, temperature, ionic strength, and solvent.

Tunable kHz Deep Ultraviolet (193–210 nm) Laser for Raman Applications

The performance characteristics of a kilohertz solid-state laser source for ultraviolet Raman spectroscopy are described. Deep ultraviolet (UV) excitation in the 193–210 nm region is provided by mixing of the fundamental and third harmonics of a Ti–sapphire laser, which is pumped by the second harmonic of a Q-Switched Nd–YLF laser. The combination of tunability, narrow linewidth, high average power, good stability, and kilohertz repetition rate makes this laser suitable for deep UV resonance Raman applications. The short pulse duration (∼20 ns) permits nanosecond time resolution in pump–probe applications. The low peak power and high data rate provide artifact-free spectra with a high signal-to-noise ratio. UV Raman cross-section and Raman excitation profiles are reported for gaseous O2 (relative to N2), aqueous ClO4−, tyrosine, phenylalanine, tryptophan, histidine, and hemoglobin excited between 193 nm and 210 nm to illustrate laser performance.