Limited sensitivity of permafrost soils to heavy rainfall across Svalbard ecosystems.

Together with warming air temperatures, Arctic ecosystems are expected to experience increases in heavy rainfall events. Recent studies report accelerated degradation of permafrost under heavy rainfall, which could put significant amounts of soil carbon and infrastructure at risk. However, controlled experimental evidence of rainfall effects on permafrost thaw is scarce. We experimentally tested the impact and legacy effect of heavy rainfall events in early and late summer for five sites varying in topography and soil type on the High Arctic archipelago of Svalbard. We found that effects of heavy rainfall on soil thermal regimes are small and limited to one season. Thaw rates increased under heavy rainfall in a loess terrace site, but not in polygonal tundra soils with higher organic matter content and water tables. End-of-season active layer thickness was not affected. Rainfall application did not affect soil temperature trends, which appeared driven by timing of snowmelt and organic layer thickness, particularly during early summer. Late summer rainfall was associated with slower freeze-up and colder soil temperatures the following winter. This implies that rainfall impacts on Svalbard permafrost are limited, locally variable and of short duration. Our findings diverge from earlier reports of sustained increases in permafrost thaw following extreme rainfall, but are consistent with observations that maritime permafrost regions such as Svalbard show lower rainfall sensitivity than continental regions. Based on our experiment, no substantial in-situ effects of heavy rainfall are anticipated for thawing of permafrost on Svalbard under future warming. However, further work is needed to quantify permafrost response to local redistribution of active layer flow under natural rainfall extremes. In addition, replication of experiments across variable Arctic regions as well as long-term monitoring of active layers, soil moisture and local climate will be essential to develop a panarctic perspective on rainfall sensitivity of permafrost.

Detection of immunoglobulin kappa light-chain gene rearrangement patterns by Southern blot analysis.

Immunoglobulin light-chain (IgL) gene rearrangements occur in a sequential order during normal B-cell differentiation with Ig kappa gene rearrangements prior to Ig lambda gene rearrangements. Therefore, Ig kappa producing B-cells usually retain Ig lambda genes in germline configuration, whereas the Ig kappa genes are generally deleted on one or both alleles in most Ig lambda producing B-cells. The deletion processes in the Ig kappa locus are mediated via rearrangement of the kappa deleting element (Kde), which is located approximately 24 kb downstream of the constant (C) kappa gene segment. Kde rearrangements can delete the C kappa region (including the Ig kappa enhancer) or the complete joining (J) kappa-C kappa region via rearrangements to a heptamer recombination signal sequence in the J kappa-C kappa intron (intron RSS), or via rearrangement to a variable (V) kappa gene segment, respectively. To improve the Southern blot detection of clonal Ig kappa gene rearrangements and deletions in B-lineage malignancies, we developed a new set of optimal J kappa, C kappa, and Kde probes, and made a detailed restriction map of the J kappa, C kappa, and Kde region. Extensive Southern blot studies revealed that rearrangements in the J kappa gene region are optimally detectable by use of a J kappa probe in combination with at least two appropriate restriction enzymes, i.e. BamHI, BglII, EcoRI, HindIII, and/or SacI. J kappa gene rearrangements are also detectable with the C kappa probe in BglII and BamHI digests, if no deletion of the C kappa region has occurred. The two different types of Kde-mediated J kappa and/or C kappa gene deletions are easily detectable with the Kde probe in BglII, HindIII and/or EcoRI digests. This is in contrast to the inaccurate information obtained with the J kappa and C kappa probes, because these probes can detect deletions only in the form of decreased densities of J kappa and/or C kappa germline bands in the absence of rearranged bands. Our detailed analysis of 217 B-lineage leukemias revealed that 62% (69/111) of precursor B-cell acute lymphoblastic leukemias had rearranged and/or deleted Ig kappa genes. All 53 Ig lambda+ chronic B-cell leukemias contained Ig kappa gene deletions; in 75% this concerned biallelic J kappa and/or C kappa gene deletions. Virtually all Ig kappa gene deletions appeared to be mediated via Kde rearrangements, while only 1.5% of the Ig kappa gene deletions were mediated via an alternative deletion mechanism which involved the J kappa region.

Differences in immunoglobulin heavy chain gene rearrangmeent patterns between bone marrow and blood samples in childhood precursor B-acute lymphoblastic leaukemia at diagnosis.

Bone marrow (BM) and corresponding peripheral blood (PB) samples from 30 patients with precursor B-acute lymphoblastic leukemia (precursor B-ALL) were analyzed for the configuration of their immunoglobulin (Ig) heavy chain (IgH) and Ig kappa chain (Ig kappa) genes. Rearrangements and/or delections of the IgH and Ig kappa genes were detected in 100 and 47% of patients in this series of precursor B-ALL, respectively. Multiple rearranged IgH gene bands, generally differing in density, were found in 10 precursor B-ALL samples. This multi-band pattern is most probably caused by subclone formation due to continuing rearrangement processes. In five of the 10 bi/oligoclonal cases (50%) differences in IgH gene rearrangement patterns between BM and PB samples were observed, which could be interpreted as the presence of an edeletections of the IgH and Ig kappa genestra subclone in two cases and differences in the size of the subclones in three cases. In the 20 monoclonal precursor B-ALL, no dissimilarities in IgH gene rearrangement patterns between BM and the corresponding PB samples were found. Differences in Ig kappa gene rearrangement patterns between BM and PB were not observed in this series of precursor B-ALL, which is in line with the finding that no multiple Ig kappa gene rearrangements were detectable. In all five cases, the edelections of the IgH and Ig kappa genestra subclones or the relatively larger sized subclones were found in the BM samples, suggesting that subclone formation in precursor B-ALL occurs in the tissue compartment from which the precursor B-ALL cells are thought to originate. This phenomenon will lead to underestimation of subclone formation, if only IgH gene analysis of PB samples is performed. In addition, it will hamper the detection of minimal residual disease by the polymerase chain reaction mediated amplification of 'leukemia-specific' IgH gene junctional regions, because it is unpredictable which subclone will cause minimal residual disease and/or relapse.

Southern blot detection of immunoglobulin lambda light chain gene rearrangements for clonality studies.

Southern blot analysis of immunoglobulin (Ig) genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig lambda (Ig lambda) gene rearrangements, we carefully determined the precise restriction map of the J-C lambda gene region, developed a suitable C lambda probe (IGLC3), and evaluated relevant restriction enzymes in combination with the IGLC3 probe. For the latter purpose, we selected 75 B cell malignancies with proven expression of Ig lambda protein chains in order to be sure that each malignancy contained at least one clonally rearranged IG lambda allele. Our extensive Southern blot analyses with the IGLC3 probe in EcoRI and/or HindIII digests revealed that combined EcoRI/HindIII digestion detected Ig lambda gene rearrangements in 95% of the 75 patients and 94% of the 98 rearranged alleles. In contrast, HindIII and EcoRI single digests allowed detection of rearrangements in only 78% and 83% of the patients, and 67% and 79% of rearranged alleles, respectively. We conclude that the use of the IGLC3 probe in combined EcoRI/HindIII digests is superior to EcoRI and HindIII single digests. This probe/enzyme combination is informative for clonality studies in approximately 95% of patients.

Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursor B-cell origin.

Sixty precursor B-cell acute lymphoblastic leukemia (ALL) patients were analyzed for the configuration of their immunoglobulin (Ig) genes. Rearrangements and/or deletions of the Ig heavy chain (IgH), Ig kappa chain (Ig kappa), and Ig lambda chain (Ig lambda) genes were detected in 98, 48, and 23% of cases, respectively. Although these percentages suggest the presence of a hierarchical order in IgH and Ig light chain (IgL) gene rearrangements during B-cell differentiation, no correlation was found between the immunophenotype of the precursor B-ALL and the arrangement patterns of their IgH and IgL genes. Multiple rearranged IgH gene bands, generally differing in density, were found in 27 (45%) of the precursor B-ALL in various restriction enzyme digests. Cytogenetic data were used to determine whether the presence of more than two rearranged IgH gene bands was caused by hyperdiploidy of chromosome 14 or other chromosome 14 aberrations. The combined cytogenetic and IgH gene data allowed the precursor B-ALL to be divided into three groups: a monoclonal group (n = 36; 60%), a biclonal group (n = 16; 27%), and an oligoclonal group (n = 8; 13%). In five biclonal ALL biclonality at the Ig kappa gene level was also found. Such subclone formation was not detected at the Ig lambda gene level. As the detection limit of the Southern blot technique is 2-5%, it might well be that small subclones remained undetected, implying that the frequency of subclone formation at the IgH gene level in precursor B-ALL is probably higher than 40%. It has been suggested that precursor B-ALL with multiple IgH gene rearrangements have a higher tendency to relapse. Although higher relapse rates were found in the oligoclonal group (53%) and in the combined bi-oligoclonal group (33%) compared with the monoclonal group (20%), the log rank trend test showed no significance. The occurrence of multiple subclones in precursor B-ALL as found by IgH gene analyses will severely hamper the detection of minimal residual disease using the polymerase chain reaction (PCR) mediated amplification of 'tumor-specific' IgH gene junctional regions, because it cannot be predicted which detectable (or undetectable) subclone will cause minimal residual disease and/or relapse. Therefore it can be expected that the PCR technique will frequently produce false negative results during the follow-up of precursor B-ALL.

Immunophenotypic changes between diagnosis and relapse in childhood acute lymphoblastic leukemia.

To get more insight into the phenotypic changes of childhood acute lymphoblastic leukemia (ALL) at relapse, a detailed morphological and immunophenotypic study in 40 childhood ALL cases (32 precursor B-ALL and 8 T-ALL) was performed. Expression patterns of non-lineage specific markers (terminal deoxynucleotidyl transferase (TdT), CD34, and HLA-DR), B-lineage markers (CD10, CD19, CD20, and CD22), T-lineage markers (CD1, CD2, CD3, CD4, CD5, CD7, and CD8), and cross-lineage myeloid markers (CD14, CD15, and CD33) were compared at diagnosis and relapse. In case of low blast counts (< or = 70%) at relapse, double labeling for membrane markers and TdT was used in order to define the precise immunophenotype of the TdT+ leukemic cells. An immunological marker-shift was defined as either a conversion from positive to negative and vice versa or a difference in positivity of > or = 50%. Morphological differences between diagnosis and relapse were detected in 34% of precursor B-ALL and 14% of T-ALL. Differences in immunological marker expression were found in 72% of precursor B-ALL and in 75% of T-ALL, and generally concerned minor shifts with loss or acquisition of a few markers. The morphological shifts and immunophenotypic shifts were not correlated. Immunophenotypic shifts were found for all markers tested in precursor B-ALL, except for HLA-DR. Shifts in CD10 expression (16% of cases) were only observed in relapses occurring 30 months or more after diagnosis. In four precursor B-ALL an intra-lineage shift was found at relapse (one common ALL to null ALL and three pre-B-ALL to common ALL or null ALL) and two precursor B-ALL cases were diagnosed as acute non-lymphocytic leukemia at relapse based on morphology and immunophenotype. In T-ALL, neither intra-lineage nor inter-lineage shifts were observed, although shifts were detected in all T cell markers tested, except for the lineage specific CD3 and T cell receptor (TcR) markers. In conclusion, immunophenotypic shifts at relapse frequently occur in precursor B-ALL and T-ALL, in a small percentage leading to an intra-lineage shift (10%) or inter-lineage shift (5%). Therefore immunophenotypic monitoring of minimal residual disease in ALL patients should be based on multiple marker combinations, preferably together with polymerase chain reaction analysis of rearranged immunoglobulin and/or TcR genes or chromosome aberrations.

Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.

Southern blot analysis of immunoglobulin (Ig) and T-cell receptor genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig heavy-chain (IgH) gene rearrangements, we carefully determined the precise restriction map of the joining (J)H and constant (C)mu region of the IgH locus, and evaluated relevant combinations of restriction enzymes with JH and C mu probes. Our extensive Southern blot analyses revealed that rearrangements in the JH region are optimally detectable by use of a JH probe in combination with at least two restriction enzyme digests which are not affected by polymorphisms, and which produce small germline bands (e.g. BglII and BamHI/HindIII), thereby reducing the chance of comigration of germline and/or rearranged bands. Application of a JH or a C mu probe in combination with BamHI or EcoRI digests should be avoided, because of the large size of the restriction fragments and the occurrence of polymorphisms. Comparison of different types of JH probes demonstrated that optimally reproducible signals, independent of the rearranged JH gene segment, are only obtained if the JH probe is complementary to the 3' flanking sequences of the JH gene region, such as our IGHJ6 probe.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vkappa gene segments (Vkappa-Kde rearrangements) or to the heptamer recombination signal sequence in the Jkappa-Ckappa intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vkappa gene family usage in the Vkappa-Kde rearrangements in our series of B-lineage leukemias was comparable to Vkappa gene family usage in functional Vkappa-Jkappa rearrangements in normal and malignant mature B cells, except for a higher frequency of VkappaII family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10(-4) to 10(-5). Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be 'end-stage' rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

Bioefficacy of beta-carotene dissolved in oil studied in children in Indonesia.

BACKGROUND: More information on the bioefficacy of carotenoids in foods ingested by humans is needed. OBJECTIVE: We aimed to measure the time required for isotopic enrichment of beta-carotene and retinol in serum to reach a plateau, the extent of conversion of beta-carotene dissolved in oil with use of beta-carotene and retinol specifically labeled with 10 (13)C atoms, and the intraindividual variation in response. DESIGN: Indonesian children aged 8--11 y (n = 35) consumed 2 capsules/d, 7 d/wk, for < or =10 wk. Each capsule contained 80 microg [12,13,14,15,20,12',13',14',15',20'-(13)C(10)]beta-carotene and 80 microg [8,9,10,11,12,13,14,15,19,20-(13)C(10)]retinyl palmitate. Three blood samples were drawn per child over a period of < or =10 wk. HPLC coupled with atmospheric pressure chemical ionization liquid chromatography-mass spectrometry was used to measure the isotopic enrichment in serum of retinol with [(13)C(5)]retinol and [(13)C(10)]retinol and of beta-carotene with [(13)C(10)]beta-carotene. The beta-carotene in the capsules used had a cis-trans ratio of 3:1. RESULTS: Plateau isotopic enrichment was reached by day 21. The amount of beta-carotene in oil required to form 1 microg retinol was 2.4 microg (95% CI: 2.1, 2.7). The amount of all-trans-beta-carotene required to form 1 microg retinol may be lower. CONCLUSIONS: The efficiency of conversion of this beta-carotene in oil was 27% better than that estimated previously (1.0 microg retinol from 3.3 microg beta-carotene with an unknown cis-trans ratio). The method described can be extended to measure the bioefficacy of carotenoids in foods with high precision, requiring fewer subjects than other methods.

Reversible migratory osteoporosis in renal oncocytoma mimicking renal cell carcinoma with bone metastases.

We report a case in which initially the wrong diagnosis of renal cell carcinoma with bone metastases was made. Nephrectomy and bone biopsy led to the right diagnosis of oncocytoma with transient osteoporosis. This report stresses the importance of pathological investigation and points to oncocytoma in the differential diagnosis of solid renal masses. In addition, the possible relationship between this tumour and migratory osteoporosis, which disappeared after surgery, is described.

Giant proximity effect in cuprate superconductors.

Using an advanced molecular beam epitaxy system, we have reproducibly synthesized atomically smooth films of high-temperature superconductors and uniform trilayer junctions with virtually perfect interfaces. We found that supercurrent runs through very thick barriers. We can rule out pinholes and microshorts; this "giant proximity effect" (GPE) is intrinsic. It defies the conventional explanation; it might originate in resonant tunneling through pair states in an almost-superconducting barrier. GPE may also be significant for superconducting electronics, since thick barriers are easier to fabricate.

No mixing of superconductivity and antiferromagnetism in a high-temperature superconductor.

There is still no universally accepted theory of high-temperature superconductivity. Most models assume that doping creates 'holes' in the valence band of an insulating, antiferromagnetic 'parent' compound, and that antiferromagnetism and high-temperature superconductivity are intimately related. If their respective energies are nearly equal, strong antiferromagnetic fluctuations (temporally and spatially restricted antiferromagnetic domains) would be expected in the superconductive phase, and superconducting fluctuations would be expected in the antiferromagnetic phase; the two states should 'mix' over an extended length scale. Here we report that one-unit-cell-thick antiferromagnetic La2CuO4 barrier layers remain highly insulating and completely block a supercurrent; the characteristic decay length is 1 A, indicating that the two phases do not mix. We likewise found that isolated one-unit-cell-thick layers of La1.85Sr0.15CuO4 remain superconducting. The latter further implies that, on doping, new electronic states are created near the middle of the bandgap. These two findings are in conflict with most proposed models, with a few notable exceptions that include postulated spin-charge separation.

Antibody L26 recognizes an intracellular epitope on the B-cell-associated CD20 antigen.

Monoclonal antibody L26 is a highly selective marker of B cells and B-cell neoplasms in paraffin-embedded tissues, but it suffers from the drawback that the target molecule has not been identified. In this paper we provide evidence by two independent techniques that antibody L26 recognizes an intracellular epitope on the CD20 antigen (a pan B-cell marker). When this antigen was redistributed on the surface of unfixed viable B cells by incubation with monoclonal anti-CD20 followed by anti-mouse Ig, the diffuse cytoplasmic staining of L26 was abolished and replaced by coincident dotlike labeling for antibody L26 and the CD20 antigen. None of the other antibodies tested (covering 10 different B-cell-associated antigens) had this effect on the L26 staining pattern. Furthermore, COS-1 cells transfected with cDNA encoding the CD20 molecule gave positive staining with antibody L26 and with two other CD20 reagents, but not with antibodies to other pan B-cell markers (eg, CD19 and CD22).

Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis.

The rearrangement patterns of Ig and T-cell receptor (TcR) genes were studied by Southern blot analysis in 30 precursor B-cell acute lymphoblastic leukemias (B-ALLs) and 10 T-ALLs at diagnosis and subsequent relapse. Eight precursor B-ALLs appeared to contain biclonal/oligoclonal Ig heavy-chain (IgH) gene rearrangements at diagnosis. Differences in rearrangement patterns between diagnosis and relapse were found in 67% (20 cases) of precursor B-ALLs (including all eight biclonal/oligoclonal cases) and 50% (five cases) of T-ALLs. In precursor B-ALLs, especially changes in IgH and/or TcR-delta gene rearrangements were found (17 cases), but also changes in TcR-beta, TcR-gamma, Ig kappa, and/or Ig lambda genes (11 cases) occurred. The changes in T-ALLs concerned the TcR-beta, TcR-gamma, TcR-delta, and/or IgH genes. Two precursor B-ALLs showed completely different Ig and TcR gene rearrangement patterns at relapse, suggesting the absence of a clonal relation between the leukemic cells at diagnosis and relapse and the development of a secondary leukemia. The clonal evolution in the other 23 ALL patients was based on continuing rearrangement processes and selection of subclones. The development of changes in Ig and TcR gene rearrangement patterns was related to remission duration, suggesting an increasing chance of continuing rearrangement processes with time. These immunogenotypic changes at relapse occurred in a hierarchical order, with changes in IgH and TcR-delta genes occurring after only 6 months of remission duration, whereas changes in other Ig and TcR genes were generally detectable after 1 to 2 years of remission duration. The heterogeneity reported here in Ig and/or TcR gene rearrangement patterns at diagnosis and relapse might hamper polymerase chain reaction (PCR)-mediated detection of minimal residual disease (MRD) using junctional regions of rearranged Ig or TcR genes as PCR targets. However, our data also indicate that in 75% to 90% of ALLs, at least one major rearranged IgH, TcR-gamma, or TcR-delta band (allele) remained stable at relapse. We conclude that two or more junctional regions of different genes (IgH, TcR-gamma, and/or TcR-delta) should be monitored during follow-up of ALL patients for MRD detection by use of PCR techniques. Especially in biclonal/oligoclonal precursor B-ALL cases, the monitoring should not be restricted to rearranged IgH genes, but TcR-gamma and/or TcR-delta genes should be monitored as well, because of the extensive changes in IgH gene rearrangement patterns in this ALL subgroup.

Southern blot patterns, frequencies, and junctional diversity of T-cell receptor-delta gene rearrangements in acute lymphoblastic leukemia.

Southern blot analysis of T-cell receptor (TCR)-delta gene rearrangements is useful for diagnostic studies on the clonality of lymphoproliferative diseases. We have developed 18 new TCR-delta gene probes by use of the polymerase chain reaction (PCR) techniques. Application of these probes for detailed analysis of the TCR-delta genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL), and 91 precursor B-ALL allowed us to determine the TCR-delta gene restriction map for five restriction enzymes, as well as the Southern blot restriction enzyme patterns of all theoretically possible TCR-delta gene rearrangements. Based on this information, it appeared that 97% of all 213 detected TCR-delta gene rearrangements in our series of ALL could be detected by use of the TCRDJ1 probe and that the majority (76%) of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V delta 1-J delta 1 (33%), V delta 2-J delta 1 (10%), and V delta 3-J delta 1 (7%) and the incomplete rearrangement D delta 2-J delta 1 (11%). In precursor B-ALL, the majority of rearrangements consisted of V delta 2-D delta 3 (72%) and D delta 2-D delta 3 (10%). The junctional diversity of these 6 preferential TCR-delta rearrangements was analyzed and showed an extensive junctional insertion (approximately 30 nucleotides) for complete V delta-J delta rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-delta gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-delta gene rearrangements, are important for TCR-delta gene studies in ALL; all TCR-delta gene rearrangements can be detected and the majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis of TCR-delta gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.

A liquid chromatography-mass spectrometry method for the quantification of bioavailability and bioconversion of beta-carotene to retinol in humans.

A method based on high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (APCI LC-MS) was developed for the quantification of the bioavailability of retinyl palmitate and beta-carotene and the bioconversion of beta-carotene to retinol in humans. Following oral administration of [8,9,10,11,12,13,14,15,19,20-13C10]-retinyl palmitate and [12,13,14,15,20,12',13',14',15',20'-13C10]-beta-carotene at physiological doses to children between 8 and 11 years of age, blood samples were drawn and serum was prepared. Retinol and beta-carotene were extracted from 0.2- and 1.0-mL serum samples, respectively, and analyzed using reversed-phase HPLC with a C30 column interfaced to an APCI mass spectrometer. Unlike other LC-MS assays for carotenoids, no additional purification steps were necessary, nor was any derivatization of retinol or beta-carotene required. APCI LC-MS showed a linear detector response for beta-carotene over 4 orders of magnitude. Using selected ion monitoring to record the elution profile of protonated circulating beta-carotene at m/z 537 and [13C10]-beta-carotene at m/z 547, the limit of detection was determined to be 0.5 pmol injected on-column. To assess the ratio of labeled to unlabeled retinol, selected ion monitoring was carried out at m/z 269, 274, and 279. These abundant fragment ions corresponded to the loss of water from the protonated molecule of circulating retinol, [13C5]-retinol (metabolically formed from orally administered [13C10]-beta-carotene), and [13C10]-retinol (formed by hydrolysis of [13C10]-retinyl palmitate). The ratios of labeled to unlabeled retinol and the ratio of labeled to unlabeled beta-carotene were calculated. Combined with standard HPLC measurement of beta-carotene and retinol concentration and a mathematical model, these results showed that this simple LC-MS method can be used to quantify beta-carotene bioavailability and its bioconversion to retinol at physiologically relevant doses.

Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: electronic structure of the retinylidene chromophore in rhodopsin.

11-Z-[8,9,10,11,12,13,14,15,19,20-(13)C10]Retinal prepared by total synthesis is reconstituted with opsin to form rhodopsin in the natural lipid membrane environment. The 13C shifts are assigned with magic angle spinning NMR dipolar correlation spectroscopy in a single experiment and compared with data of singly labeled retinylidene ligands in detergent-solubilized rhodopsin. The use of multispin labeling in combination with 2-D correlation spectroscopy improves the relative accuracy of the shift measurements. We have used the chemical shift data to analyze the electronic structure of the retinylidene ligand at three levels of understanding: (i) by specifying interactions between the 13C-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schiff base in rhodopsin, and (iii) by evaluating the total charge on the carbons of the retinylidene chromophore. In this way it is shown that a conjugation defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative chemical shifts at the odd-numbered carbons (Delta(sigma)odd) of 11-Z-protonated Schiff base models relative to the unprotonated Schiff base can be used to measure the extent of delocalization of positive charge into the polyene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delta(sigma)odd appears to correlate linearly with the frequency of maximum visible absorption. Since rhodopsin has the largest value of Delta(sigma)odd, the data contribute to existing and converging spectroscopic evidence for a complex counterion stabilizing the protonated Schiff base in the binding pocket.