Karen P. Peterson

The prenatal detection of significant alcohol exposure with maternal blood markers

OBJECTIVE To examine the efficacy of a combination of 4 blood markers of alcohol use in detecting alcohol-abusing pregnant women. STUDY DESIGN Two new markers of alcohol use, whole blood-associated acetaldehyde and carbohydrate-deficient transferrin, and 2 traditional markers of alcohol use, gamma-glutamyl transpeptidase and mean red blood cell volume, were measured in the blood of pregnant women. Each woman was interviewed about alcohol and drug use, medical and obstetric histories, and nutrition. Each infant was examined by a clinician who was blinded to exposure status. RESULTS All of the women who reported drinking an average of 1 or more ounces of absolute alcohol per day had at least 1 positive blood marker. The infants of mothers with 2 or more positive markers had significantly smaller birth weights, lengths, and head circumferences than the infants with negative maternal screens. The presence of 2 or more positive markers was more predictive of infant outcome than any self-reporting measure. CONCLUSIONS These markers, which detect more at-risk pregnant women than self-reporting methods, could lead to better efforts at detection and prevention of alcohol-induced fetal damage.

Alcohol Metabolism in Asian-American Men with Genetic Polymorphisms of Aldehyde Dehydrogenase

Epidemiologic studies have found that rates of alcohol use and alcoholism in persons of Asian descent are lower than rates in other ethnic groups. One possible reason is that about half of certain Asians, including Chinese, Japanese, and Korean persons, have a deficiency of the low-Km mitochondrial aldehyde dehydrogenase (ALDH2) isoenzyme, which is responsible for metabolizing acetaldehyde. A deficiency of ALDH2 results from inheritance of the mutant ALDH2*2 allele, a dominant mutation that exerts its effect both by reducing enzyme activity and increasing the turnover of this activity [1, 2]. After ingestion of alcohol, the faces of Asians with one or both alleles of ALDH2*2 become visibly flushed. Asians who are homozygous for ALDH2*1 generally lack visible alcohol-induced flushing or experience only a mild flush response. The dominance of the ALDH2 mutation, however, does not seem to be complete; phenotypic differences are associated with the three ALDH2 genotypes. Asians who are homozygous for ALDH2*2 drink very little alcohol [3], and no studies have found alcoholic persons with this genotype [4-8]. Asians who are heterozygous for ALDH2*2 drink less alcohol and are also less likely to be alcoholic compared with Asians with ALDH2*1 alleles, but they are not fully protected from alcoholism. Approximately 12% of alcoholic Asians have the ALDH2*1/2*2 genotype [5]. In the context of alcoholism or lower alcohol intake, Asian persons who are heterozygous for ALDH2*2 may be more vulnerable to alcohol-associated conditions, including liver disease [6-9], asthma [9], and esophageal cancer [10]. The three ALDH2 genotypes are also associated with variability in response to alcohol [11]. Asians who are homozygous for ALDH2*2 are very sensitive to alcohol and have tachycardia, hypotension, and vomiting after ingesting a moderate amount of alcohol. Asians who are heterozygous for ALDH2*2 are more sensitive to alcohol than Asians with ALDH2*1 alleles, although the response of the former is not necessarily aversive. Among Asians with an ALDH2 deficiency, differences in sensitivity to alcohol may be mediated by differences in alcohol metabolism, slower elimination of alcohol, or accumulation of acetaldehyde in the blood [12]. Some studies [13-17] have measured blood levels of alcohol or acetaldehyde after ingestion of alcohol in Asians who were known to have ALDH2 genotypes, but these studies had an inadequate sample size, did not include a placebo control, or did not control for use of alcohol and cigarettes (which can alter alcohol metabolism). We measured blood levels of alcohol and acetaldehyde after ingestion of alcoholic or placebo beverages in Asian-American men who underwent genotyping at the ALDH2 locus. Particular attention was given to matching the groups for age, height, weight, history of alcohol use, and history of smoking. Methods Asian-American men 21 to 25 years of age were recruited from advertisements in university newspapers for our randomized, double-blind, crossover study. They completed a questionnaire that solicited information on demographic characteristics; patterns of and problems with alcohol and drug use; and family history of alcohol, drug, and psychiatric problems. We excluded persons who completely abstained from alcohol, persons who had consumed more than 60 standard alcoholic drinks per month during the previous 6 months, and persons who reported that either biological parent was not of Chinese, Japanese, or Korean descent. Thirty-five men who did not have a personal or family history of alcohol dependence and who had no evidence of other substance dependence, major psychiatric disorders, or medical disorders gave informed consent to participate in two test sessions. The study was approved by the institutional review board at the Scripps Clinic and Research Foundation. Participants were asked to refrain from using alcohol, cigarettes, and other drugs (including aspirin, nonsteroidal anti-inflammatory agents, and anti-histamines) that might alter alcohol metabolism for 3 days before testing. On test days, each participant arrived at the clinic at 7:30 a.m. after an overnight fast. He then ate a low-fat breakfast (two slices of dry toast and juice), and an indwelling heparin lock was inserted for drawing blood. At the first session, blood was drawn and genotyping at the ALDH2 locus was done by using polymerase chain reaction of DNA and allele-specific oligonucleotide probes [1]. At 9:00 a.m., each participant was given a placebo beverage (3 mL of 95% alcohol in a reservoir on top of noncaffeinated, sugar-free soda) or 0.75 mL of 95% alcohol (0.56 g/kg of body weight) as a 20%-by-volume solution in the same mixer. The alcohol and placebo were ingested over 7 minutes through a placebo alcohol apparatus [18]. Blood was drawn to determine levels of alcohol and acetaldehyde before beverage ingestion and 15, 30, 45, 60, 90, 120, and 150 minutes after beverage ingestion. Blood alcohol concentrations were determined by using a modified alcohol dehydrogenase assay [19]. The rate of alcohol elimination (mg/kg per hour) was calculated from the slope of the pseudolinear decline of the blood alcohol concentration-time curve (usually from the 90-, 120-, and 150- minute samples) by using linear least-squares regression. Blood acetaldehyde levels were determined by using a modified fluorigenic high-performance liquid chromatographic assay [20] that had a detection sensitivity in the picomole range and intra-assay and interassay precisions of 2.4% and 3.7%, respectively. Statistical analyses, done by using SYSTAT software (SYSTAT, Inc., Evanston, Illinois), focused on differences between participants with ALDH2*1/2*1 and those with ALDH2*1/2*2. Demographic information, data on recent alcohol and cigarette use, peak blood alcohol concentration, time to peak blood alcohol concentration, volume of distribution, and rate of alcohol elimination were analyzed by using one-way analysis of variance; ALDH2 genotype was a between-participant variable. Data on blood alcohol concentration and acetaldehyde level were analyzed by using separate 2 8 analysis of variance for alcohol and placebo sessions; ALDH2 genotype was a between-participant variable, and time was a repeated measurement. Significant interactions were then analyzed by using post hoc comparisons with contrast matrices. The Bonferroni correction was used to limit the familywise error rate to 0.05 for comparisons among the placebo session time points and among the alcohol session time points. Results Genotyping for ALDH2 revealed 20 participants who had ALDH2*1/2*1 genotype, 13 who had ALDH2*1/2*2 genotype, and 2 who had ALDH2*2/2*2 genotype. Three participants (1 with ALDH2*1/2*2 genotype and the 2 with ALDH2*2/2*2 genotype) became ill after ingesting alcohol and were excluded from analyses because of missing data. One participant with ALDH2*1/2*2 genotype whose acetaldehyde levels exceeded 4 SDs from the mean (most likely as a result of instrumentation error) was also excluded from data analyses. The Table 1 shows demographic information and patterns of recent alcohol and cigarette use for the remaining 31 men. The ALDH2 genotype groups did not differ significantly for any of these variables; this reflects participant selection. Table 1. Demographic Information and Recent Patterns of Alcohol and Cigarette Use in 20 Asian-American Men with ALDH2*1/2*1 Genotype and 11 Asian-American Men with ALDH2*1/2*2 Genotype* Mean peak blood alcohol concentration SD was 81.3 12.48 mg/dL; the peak occurred 43.1 15.85 minutes after ingestion of alcohol. Mean volume of distribution was 0.718 0.1124 L/kg of body weight, and the mean rate of alcohol elimination was 97.8 33.97 mg/kg per hour. The ALDH2 genotype groups did not differ significantly for any of these variables. Mean blood alcohol concentrations for the alcohol session and mean acetaldehyde levels for the placebo and alcohol sessions, measured over time according to ALDH2 genotype, are shown in the (Figure 1). Figure 1. Mean (SD) blood levels of alcohol and acetaldehyde before and after ingestion of a placebo beverage containing 3 mL of 95% alcohol and an alcoholic beverage (0. n n P Analysis of variance revealed that the main effects for ALDH2 genotype and the interaction between ALDH2 genotype and time were not significant for the data on blood alcohol concentration from the alcohol session. Analysis of variance also revealed that the main effects for ALDH2 genotype and the interaction between ALDH2 genotype and time were significant for the data on acetaldehyde levels from the placebo sessions (ALDH2 genotype, P < 0.005; interaction between ALDH2 genotype and time, P < 0.013) and the alcohol sessions (ALDH2 genotype, P < 0.002; interaction between ALDH2 genotype and time, P < 0.001). Post hoc analyses with Bonferroni corrections revealed significant group differences 30, 45, and 60 minutes after placebo ingestion and 60, 90, 120, and 150 minutes after alcohol ingestion. Discussion We found that Asian-American men who were heterozygous for the ALDH2*2 allele did not differ from carefully matched men who were homozygous for the ALDH2*1 allele in measures of blood alcohol concentration overall or at any time after alcohol ingestion. These findings are consistent with the results of one study [14] but differ from those of other studies [15-17] in which persons with ALDH2*2 alleles had significantly slower rates of alcohol elimination than did persons with ALDH2*1 alleles. These discrepancies may result from group differences on important variables, especially history of alcohol use, that we controlled for. We also found that, despite equivalent blood alcohol concentrations, participants with ALDH2*1/2*2 genotype had significantly higher blood acetaldehyde levels after ingesting the alcohol beverage than did participants with ALDH2*1/2*1 genotype. These findings suggest that blood acetaldehyde levels rather than bloo

Effect of hemoglobin concentration variation on the accuracy and precision of glucose analysis using tissue modulated, noninvasive, in vivo Raman spectroscopy of human blood: a small clinical study

Tissue modulated Raman spectroscopy was used noninvasively to measure blood glucose concentration in people with type I and type II diabetes with HemoCue fingerstick measurements being used as reference. Including all of the 49 measurements, a Clarke error grid analysis of the noninvasive measurements showed that 72% were A range, i.e., clinically accurate, 20% were B range, i.e., clinically benign, with the remaining 8% of measurements being essentially erroneous, i.e., C, D, or E range. Rejection of 11 outliers gave a correlation coefficient of 0.80, a standard deviation of 22 mg/dL with p<0.0001 for N=38 and places all but one of the measurements in the A and B ranges. The distribution of deviations of the noninvasive glucose measurements from the fingerstick glucose measurements is consistent with the suggestion that there are at least two systematic components in addition to the random noise associated with shot noise, charge coupled device spiking, and human factors. One component is consistent with the known variation of fingerstick glucose concentration measurements from laboratory reference measurements made using plasma or whole blood. A weak but significant correlation between the deviations of noninvasive measurements from fingerstick glucose measurements and the test subjects hemoglobin concentration was also observed.

Silica Sol-Gel Encapsulation of Pancreatic Islets:

Abstract We developed a biocomposite material containing silica ceramic. The sol-gel technology in which ceramic materials are polymerized from liquid solutions at room temperature and physiologic pH can be used to produce ceramics that have a determined pore size and that contain living organisms or cells. Capsules were stable to extreme acid and base conditions as well as to trypsin in vitro for 6 months. We used insulin-secreting murine islet cells as the first mammalian material for encapsulation. Two approaches to generating successful encapsulation of islets were used: drop-tower sphere generation and emulsion. Sphere diameters of less than 1 mm were associated with positive insulin secretory capacity as documented by a static batch incubation technique. Average pore sizes were 161 Å for drop-tower spheres and 105 Å for emulsion spheres. Capsules allowed the passage of insulin and cytokines but not the passage of antibody. Implantation of encapsulated islets did not result in fibrosis of the capsule in vivo, and retrieval of capsules after 1 month in vivo documented continued insulin secretory capacity. Further in vivo experiments documented increased survival of transplant recipients despite failure to achieve normoglycemia in all but a few cases. Silica sol-gel encapsulation provides a potentially useful alternative for encapsulation of cells for transplantation or drug delivery, and further work is warranted to develop this potentially useful approach for the treatment of diabetes mellitus.

Reactivation of type 1 diabetes in patients receiving human fetal pancreatic tissue transplants without immunosuppression.

BACKGROUND Type 1 diabetes is a cell-mediated autoimmune disease. Successful transplantation of human fetal pancreatic tissue into type 1 diabetic patients must address both autoimmunity and allograft rejection. We investigated whether humoral and cellular responses to islet antigens could be demonstrated in the peripheral blood of type 1 diabetic subjects receiving human fetal pancreatic tissue transplants. METHODS We investigated peripheral blood mononuclear cell (PBMC) responses, using cellular immunoblotting, and autoantibody responses to islet proteins, before transplant and at 3-month intervals after transplant, of nine long-term type 1 diabetes patients (mean disease duration of 21 years) receiving human fetal pancreatic tissue subcutaneously into the abdominal wall without immunosuppression. RESULTS Before transplant, all nine subjects were islet cell autoantibody (ICA)-negative and seven out of nine subjects were glutamic acid decarboxylase autoantibody (GADAb)-positive. After transplant, all subjects became ICA(+) and the two patients who were GADAb(-) before transplant became GADAb(+) after transplant. Maximum PBMC reactivity to separated human fetal pancreatic proteins was observed in four patients 3 months after transplant, in one patient at 6 months, in two patients at 9 months, and in one patient at 12 months after transplant. One subject, who had PBMC reactivity to multiple islet proteins before transplant, continued to respond to multiple islet proteins throughout the study. CONCLUSIONS We conclude that the development in the peripheral blood of ICA, GADAb, and PBMC reactivity to human fetal pancreatic proteins in the trans plant recipients is most consistent with reactivation of the type 1 diabetes disease process.

Progress in the noninvasive in-vivo tissue-modulated Raman spectroscopy of human blood

We have recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. New clinical data from individuals and groups completely reproduce and extend all the earlier results. These new data reveal how certain small differences between individuals result in some variability in their noninvasive quantitation. We show the origin of this variability and how to obtain quantitative corrections based entirely on the individual measurement and tabulated data.

Noninvasive blood analysis by tissue-modulated NIR Raman spectroscopy

We present tissue modulated Raman spectroscopy as a technique for noninvasively measuring the concentration of blood analytes in vivo. We present preliminary data used to determine the best methods for analyzing our data. These experiments provide additional proof that we are indeed able to obtain the spectra of human blood in vivo and noninvasively. We discuss differences between our spectra and spectra of bulk blood in vitro. We also discuss the variations between individuals and the impact of those variations on our noninvasive blood glucose measurements.

Dapsone Decreases the Cumulative Incidence of Diabetes in Non-Obese Diabetic Female Mice

Abstract Dapsone (4,4′-diaminodiphenyl sulfone) has a large clinical experience due to its antimicrobial effects against Mycobacterium leprae, the causative agent of leprosy, and is used clinically where inflammation mediated by neutrophils is perceived to play a role. We administered dapsone in two concentrations (0.001% and 0.0001% w/w of diet) to 30 female non-obese diabetic (NOD) mice to explore the effect of dapsone on the development of IDDM following either a 1-week pulse or 20 weeks of continuous oral dapsone administration. Those mice receiving either the high or low doses of dapsone in the continuous group had a significantly reduced cumulative percentage of onset of IDDM. One of the seven mice given 0.0001% dapsone became diabetic (age 25 weeks), while none of the eight high dose (0.001%) mice developed the disease. Histological examination of pancreatic sections revealed islet infiltration in all groups of animals. The pulse and continuous experiments showed no statistically significant difference in the frequency or severity of lymphocytic infiltration. Dapsone administration did not inhibit growth, and growth rates were greater in those animals receiving the higher dapsone dose compared with the lower dose comparable to controls. We studied whether dapsone influenced murine lymphocyte function in addition to the published effects of the drug on neutrophils. At doses approximating those achieved in vivo (0.4 and 2 μg/ml), dapsone was found to inhibit murine splenocyte IL-2 and IL-4 secretion in response to concanavalin A. In view of the wide clinical experience with dapsone, randomized trials of the drug in new onset diabetes may be warranted.

Noninvasive in-vivo tissue-modulated Raman spectroscopy of human blood: microcirculation and viscosity effects

We recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and then normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. A more careful comparison of tissue modulated spectra with static in vitro and invasive in vivo spectra suggests that there are small microcirculation differences between individuals resulting in some variability in their noninvasive quantitation. This variability is based on the mechanism for blood volume normalization and various means for obtaining necessary corrections are suggested. We present new clinical data from individuals and groups supporting this mechanism and suggesting how such measurements might also be used to quantify various microcirculation abnormalities.

Peripheral blood mononuclear cell responses from type 1 diabetic patients and subjects at-risk for type 1 diabetes to human fetal pancreatic tissue proteins

BACKGROUND Fetal pancreatic tissue has been suggested to be less immunogenic than adult islets. Thus, transplantation of human fetal pancreatic tissue as treatment for type 1 diabetes has been gaining interest. To investigate this question, we tested the peripheral blood mononuclear cell (PBMC) responses from different subject populations to human adult islet proteins (AIP) versus human fetal pancreatic proteins (FPP). METHODS PBMC responses to FPP and AIP from normal controls (n=14), newly diagnosed type 1 diabetes patients (n=5), long-term type 1 diabetes patients (n=9), and subjects at-risk for development of type 1 diabetes (n=3) were studied. RESULTS We observed that normal controls demonstrated PBMC reactivity to 0-3 molecular weight regions (mwr) for both the AIP (mean+/-SD, 0.8+/-1.1) and the FPP (0.6+/-0.7). In contrast, newly diagnosed type 1 diabetic patients (<1 year) demonstrated PBMC responses to 9-16 mwr for the AIP (12.8+/-2.5) and 0-14 mwr for the FPP (6.8+/-5.0). The PBMCs from long-term type 1 diabetes patients (> 3 years) were responsive to 2-11 mwr for AIP (6.0+/-2.8) and 0-11 mwr for FPP (4.9+/-4.0). Three nondiabetic ICA positive subjects at-risk for development of type 1 diabetes demonstrated positive PBMC reactivity to 9-18 mwr for the AIP (12.7+/-3.9) and 4-18 mwr for the FPP (10.0+/-5.9). CONCLUSIONS We conclude that human fetal pancreatic proteins are not significantly less stimulatory than human adult islet proteins to PBMCs of subjects with or at risk for type 1 diabetes.