Circadian distribution of iron and ferritin in serum of healthy and type 2 diabetic males.

AIM: We examined the circulating levels of iron and ferritin in serum of seven healthy and three insulin non-dependent diabetic (Type 2) males in order to compare their circadian characteristics. METHODS: Blood samples were collected every 3h over a 24h period and were analyzed for serum iron and ferritin. RESULTS: The mean Fe level was significantly higher in healthy than in diabetic subjects: 80.0 +/- 3.3 vs. 63.0 +/- 3.7 microg/dL. The ferritin level was significantly lower in healthy than in diabetic men: 79.8 +/- 4.7 vs. 186.3 +/- 110.5 microg/L. A significant (p < 0.001) time-effect was found by ANOVA and circadian rhythm was detected at p < 0.001 in all data sets when a 24h cosine was fitted to the normalized data. Acrophases were located in mid to late morning for Fe (11:30, vs. 09:22h) and for ferritin (11:10 vs. 11:46h). DISCUSSION: We concluded that there is significant circadian variation in both serum Fe and ferritin, with predictable peaks in the mid to late morning.

Altered circadian relationship between serum nitric oxide, carbon dioxide, and uric acid in multiple sclerosis.

The free radical nitric oxide (NO*) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO-), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO- and CO2, is generally low in MS patients. We investigated the relationship between serum ONOO-, CO2, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO* metabolites nitrite/ nitrate (NO), CO2, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO2, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24h pattern in serum ONOO- formation and UA may either contribute to or reflect the disease processes in MS.

Day-night variations in blood levels of nitric oxide, T-TFPI, and E-selectin.

Circadian (8/24 hours) variations in serum nitric oxide (NO), total tissue factor pathway inhibitor (T-TFPI). and E-selectin levels were studied in healthy adults and in subjects with type II diabetes. We postulated a possibility a functional relationship between them because vascular endothelium is the primary site of their synthesis and functions. NO is released by the action of eNO synthase isoform and modulates physiologic responses (e.g., vascular dilation, relaxation, increasing blood flow, inhibition of platelet and white blood cell adhesion); T-TFPI, a coagulation inhibitor, is also released from endothelial cells, and is bound to plasma lipoproteins and to glycosaminoglycans; E-selectin is expressed on endothelial cells after activation by inflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha) and elevated levels have been reported in a variety of pathologic conditions, including diabetes. We found that obese diabetic subjects had greater mean concentrations of NO and E-selectin than healthy men, 39.25 versus 12.71 microM and 81.51 versus 26.03 ng/mL, respectively. The T-TFPI levels were essentially similar in both groups of men, 47.10 versus 48.76 ng/mL. We observed that the time of peak concentrations of T-TFPI and E-selectin was similar to the timing of NO trough levels, suggesting a possible functional relationship. It may be hypothesized, therefore, that the higher concentrations of NO, unbalanced by increases in T-TFPI and E-selectin, may result in increased vascular wall uptake of lipoproteins in diabetic subjects, who are at greater risk than healthy men for developing diffuse atherosclerosis.

Circadian variation of serum leptin in healthy and diabetic men.

Leptin, from the Greek leptos, meaning thin (in reference to its ability to reduce body fat stores), is a hormone secreted primarily by adipocytes. At one time, leptin was portrayed as a potential means of combating obesity. Recently, leptin has been identified as a potent inhibitor of bone formation, acting through the central nervous system. Since numerous studies clearly show that bone remodeling is circadian rhythmic with peak activity during sleep, it is of interest to explore circadian variability in serum leptin. Accordingly, circadian characteristics of serum leptin were examined in 7 clinically healthy men and 4 obese men with type II diabetes. Blood samples were collected for 24 h at 3 h intervals beginning at 19:00. The dark (sleep) phase of the light-dark cycle extended from 22:30 to 06:30, with brief awakening for sampling at 01:00 and 04:00. Subjects consumed general hospital meals (2400 calories) at 16:30, 07:30, and 13:30. Serum leptin levels were determined by a R&D Systems enzyme immunoassay technique. Data were analyzed by linear least-squares estimation using the population multiple components method. A statistically significant (P < .018) circadian rhythm modeled by a single 24 h cosine curve characterized the data of each group. The 24 h mean leptin level was statistically greater (P < .001) in the obese diabetic men than in the healthy men (9.47 +/- 0.66 ng/mL vs. 24.07 +/- 1.71 ng/mL, respectively). Higher leptin levels occurred between midnight and roughly 02:30, and lowest leptin levels occurred between noon and the early afternoon. The phasing of this rhythm is similar to the circadian rhythm in bone remodeling previously described. Our results suggest the findings from a single morning blood sampling for leptin may be misleading since it may underestimate the mean 24 h and peak concentrations of the hormone.

Circadian rhythm of serum total homocysteine in men.

Serum homocysteine levels were examined in a 24-hour study of 7 healthy and 5 diabetic men, revealing a statistically significant circadian rhythm (p = 0.030), normal concentrations of 11.83 +/- 1.2 vs 12.99 +/- 1.2 micromol/L, with peak values occurring during the evening (10:37 P.M.) and lowest levels occurring during the morning. These findings imply that increased atherosclerotic risk in insulin-resistant diabetics during morning hours does not appear to be explained by differences in homocysteine levels in the normal population.

Relation between circadian patterns in levels of circulating lipoprotein(a), fibrinogen, platelets, and related lipid variables in men.

BACKGROUND: A correlation has been reported between lipoprotein(a) [Lp(a)] concentration and risk for coronary artery disease. High concentrations of Lp(a) might be markers for vascular or tissue injury or might be associated with other genetic or environmental factors that can cause acute myocardial infarction. METHODS: We measured the circadian characteristics of circulating Lp(a), fibrinogen, platelets, cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol for a group of adult male volunteers who had no clinical symptoms. We obtained samples every 3 hours around the clock to assess the normal degree of variation within a 24-hour period and to test for similarities in circadian patterns and correlations with level of Lp(a). RESULTS: Each variable displayed a highly significant circadian rhythm. Lp(a), fibrinogen, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol peaked in the morning. Cholesterol and platelets peaked in the late afternoon, and triglycerides peaked in the evening. CONCLUSIONS: Although peak levels of Lp(a) and fibrinogen coincide with reported morning peak frequencies of myocardial infarction and stroke, the platelet peak appears to coincide with late afternoon peak frequencies of sudden cardiac death and fatal stroke. The data suggest that proper timing of single samples may improve the usefulness and accuracy of diagnosis, risk assessment, and therapy.

Circadian interrelationships among levels of plasma fibrinogen, blood platelets, and serum interleukin-6.

Circadian (24 h) rhythms of fibrinogen, interleukin-6 (IL-6), and platelet levels were studied in 11 males ages 46 to 72 years. Since there is a known circadian rhythm for fibrinogen and IL-6, we postulated that the peak level (acrophase) of fibrinogen would follow the acrophase of IL-6, based on the fact that IL-6 is the stimulus for fibrinogen production in the liver. Platelet levels were measured to show whether there was any correlation with the IL-6 acrophase because it has been reported that IL-6 affects megakaryocytes and platelets in dogs. We found that the acrophase for IL-6 occurred at 02:03 h and the acrophase for fibrinogen occurred at 09:16 h. Platelet counts peaked at 16:56 h. Thus, there was a positive correlation between IL-6 and fibrinogen acrophases and a negative correlation of each with the acrophase for platelets. The positive linkage of IL-6 with fibrinogen in this study suggests that suppression of IL-6 production would lower those peak fibrinogen levels that occur in the morning in association with arterial ischemic events. This could result in fewer arterial ischemic events, especially in the morning.

Circadian relationships between circulating atrial natriuretic peptides and serum calcium and phosphate in healthy humans.

Long-acting natriuretic peptide (LANP), vessel dilator (VSDL), and atrial natriuretic factor (ANF) consisting of amino acids (aa) 1 to 30, 31 to 67, and 99 to 126, respectively, of the 126-aa ANF prohormone circulate in humans. Among the biologic properties of these peptides is the ability of ANF to decrease intracellular calcium concentrations. To determine if atrial natriuretic peptides are directly related to serum calcium and/or phosphate in healthy normocalcemic humans, we examined 21 24-hour profiles of VSDL, LANP, ANF, and serum calcium and phosphate in 14 healthy humans. VSDL, LANP, and ANF each had significant (P < .001) circadian rhythms, with peak concentrations late during sleep (at 4:00 AM) being nearly twice the concentrations in the afternoon and evening. Serum calcium and phosphate also had significant circadian rhythms (P < .001) with troughs nearly opposite to those of the atrial natriuretic peptides, suggesting that atrial peptides may be important in the modulation of the circadian rhythms of calcium and phosphate. The nearly identical circadian rhythms of the atrial natriuretic peptides and of parathyroid hormone (PTH) reported by others, along with evidence that PTH may increase atrial peptide release, suggest that some of the effects attributed to PTH may be mediated by atrial natriuretic peptides.

Circadian relationships between circulating atrial natriuretic peptides and serum sodium and chloride in healthy humans.

Long-acting natriuretic peptide (LANP), vessel dilator (VSDL), and atrial natriuretic factor (ANF) consisting of amino acids 1-30, 31-67, and 99-126 of the 126 amino acid ANF prohormone, respectively, circulate in humans and have potent natriuretic properties. To determine whether these peptides have a direct relationship to serum Na and/or Cl, we examined 21 24-hour profiles of these peptides and Na and Cl in 14 healthy humans. LANP, VSDL, ANF, and Cl had significant (p < 0.001) circadian rhythms with peak concentrations at 04.00 h. The circadian rhythm of serum Na was exactly opposite. Sodium correlated negatively with LANP (p = 0.021) and ANF (p = 0.007), while Cl correlated positively with LANP (p = 0.003) and VSDL (p = 0.001). These data suggest that the atrial peptides may be important for the maintenance of serum Na and Cl within their normal ranges and in the modulation of their daily circadian rhythms.

Blood pressure and atrial natriuretic peptides correlate throughout the day.

Vessel dilator consisting of amino acids (a.a.) 31-67 and atrial natriuretic factor (ANF) composed of a.a. 99-126 of the 126 a.a. ANF prohormone circulate in humans and have potent vasodilatory properties. To determine whether these atrial natriuretic peptides are directly related to blood pressure in healthy normotensive humans, we recently had the unique opportunity to examine the circadian rhythms of vessel dilator, ANF, and blood pressure in seven individuals in 1988 and again in 1993. The changes in mean arterial pressure and systolic and diastolic blood pressure in these individuals during this 5-year hiatus allows comparison in the same individual, if circulating concentrations of atrial natriuretic peptides directly correlate with naturally occurring changes in blood pressure. In both 1988 and in 1993 vessel dilator and ANF each had significant (p < 0.001) circadian rhythms with their peak concentrations at 4:00 AM being nearly twice their concentrations at 4:00 PM. Mean arterial pressure, systolic blood pressure, and diastolic blood pressure also had significant circadian rhythms with peaks and troughs that were exactly opposite to those of ANF and vessel dilator. A significant inverse correlation between 24-hour averages of mean arterial blood pressure and 24-hour averages of vessel dilator (p = 0.05) and ANF (p = 0.02) was also found. These data suggest that vessel dilator and ANF are important for the maintenance of blood pressure within the normotensive range.

Temporal (circadian) and functional relationship between atrial natriuretic peptides and blood pressure.

Long-acting natriuretic peptide, vessel dilator, and atrial natriuretic factor consisting of amino acids (a.a.) 1 to 30, 31 to 67, and 99 to 126 of the 126-a.a. atrial natriuretic factor (ANF) prohormone, respectively, circulate in humans and have potent vasodilatory properties. To determine if these atrial natriuretic peptides are directly related to blood pressure in clinically healthy normotensive humans, we obtained 24-h profiles of vessel dilator, long-acting natriuretic peptide, ANF, and blood pressure in 10 men in 1988 and 11 men in 1993 (seven men were studied twice) to compare circulating concentrations of atrial natriuretic peptides with naturally occurring changes in blood pressure. Overall, vessel dilator, long-acting natriuretic peptide, and ANF each had significant (p<0.001) circadian rhythms, with peak concentrations late during sleep (at 04:00 h) being nearly twice their concentrations in the afternoon and evening. This high-amplitude circadian change allowed for the refinement of normal limits for ANF peptides by computing 3-hourly tolerance intervals (chronodesms) against which to compare time-specified single samples for normality. Systolic, diastolic, and mean arterial blood pressure also had significant circadian rhythms (p<0.001) with peaks and troughs that were exactly opposite those of the ANF peptides. In addition to this inverse temporal relationship, there was a significant inverse correlation between absolute values for blood pressure and each ANF peptide (p<0.001), implying a functional relationship. These data suggest that in addition to other well-established neurochemical factors, the ANF peptides (vessel dilator, long-acting natriuretic peptide, and ANF) are important for the maintenance of blood pressure and modulation of its circadian rhythm.

A quest for the relief of atherosclerosis: potential role of intrapulmonary heparin--a hypothesis.

Recent progress in the treatment of coronary artery disease is reviewed from the standpoint of changes in lifestyle, surgical techniques to revascularize the myocardium and a variety of medical interventions. Among the medical modalities, heparin appears to have a greater potential than any other agent tested to neutralize the atherogenic process at most of its stages. This potential is supported by success in clinical trials of heparin administered by intravenous, subcutaneous, pulmonary, sublingual and topical routes. The suggested self-administration of low-dose heparin by inhalation appears to be well justified and easily adaptable to home therapy. The summarized evidence suggests the need for further clinical trials to test the use of heparin in the prophylaxis of atherosclerotic disease.

A major gene for primary hypoalphalipoproteinemia.

Sixteen kindreds were ascertained through probands clinically determined to have primary hypoalphalipoproteinemia, characterized by bottom decile high-density lipoprotein cholesterol (HDL-c), but otherwise normolipidemic. Age- and sex-adjusted, standardized HDL-c levels on 64 individuals in 14 nuclear families in which the proband was a parent were analyzed using the unified mixed model of segregation analysis as implemented in the computer program POINTER. The analysis proceeded by using the likelihood of offspring conditional on the parental phenotypes (conditional likelihood), which appears to overcome the limitation of possible heterogeneity in the selection criteria and provides an appropriate correction for the ascertainment. In these families, the multifactorial contribution to the phenotype appears to be small and significant only in the offspring generation. Although it was not possible to resolve the dominance pattern at the major locus since none of a recessive, additive, or dominant hypothesis could be firmly rejected, these families provided clear evidence for a major gene. Genetic heterogeneity is still a possibility, even within "primary" hypoalphalipoproteinemia.

Primary and familial hypoalphalipoproteinemia.

Our specific aim was to assess within-family clustering of high-density lipoprotein cholesterol (HDLC) levels in kindreds identified through probands with primary hypoalphalipoproteinemia, and to determine whether, and to what degree, familial aggregation of HDLC less than or equal to the tenth percentile represents a heritable trait, familial hypoalphalipoproteinemia. Our probands were selected arbitrarily by virtue of HDLC less than or equal to the age-sex-race-specific tenth percentile as the sole dyslipoproteinemia, with an additional requirement that they be normotriglyceridemic (triglyceride levels less than the 90th percentile). The probands were also required to have primary hypoalphalipoproteinemia, not secondary to diseases and/or drugs. Fifteen of the 16 probands were men; 12 were referred because of premature myocardial infarction, angina, or stroke, 2 because of family history of premature myocardial infarction or stroke, and 2 because of low HDLC observed on routine health examinations. Two of the 16 kindreds exhibited three-generation vertical transmission of bottom decile HDLC. In three kindreds, there was also three-generation vertical transmission of bottom decile HDLC, but top decile triglycerides accompanied bottom decile HDLC in one or more generations. Eight kindreds displayed two-generation vertical transmission of bottom decile HDLC. After excluding probands, there were 11 critical matings (bottom decile HDLC by normal), with 30 living offspring, all of whom were sampled. Of these 30 offspring, 13 had bottom decile HDLC, 17 had HDLC greater than tenth percentile. The ratio of offspring with bottom decile HDLC to those of HDLC greater than tenth percentile was 13:17 (0.76/1), not significantly different from the ratio of 1/1, the ratio predictive of a dominant trait, X2(1) = 0.53, P greater than 0.4. The nearly 1:1 segregation ratio for the group of offspring was not due to the aggregation of sibships with, in general, most of the sibs, or none of the sibs affected; within-family expression of low HDLC was also not sex-linked. The 13 hypoalphalipoproteinemic offspring of 11 critical matings included only two subjects whose bottom decile HDLC was accompanied by top decile triglyceride. Our data suggests that not only (by selection) was low HDLC in the probands the sole dyslipoproteinemia, but that the segregation of low HDLC in offspring of critical matings was primarily accounted for by isolated low HDLC, not by hypoalphalipoproteinemia secondary to hypertriglyceridemia. Familial hypoalphalipoproteinemia is a heritable disorder with a pattern of transmission not significantly different from that expected by a hypothesis of mendel

A genetic study of hypoalphalipoproteinemia.

Complex segregation analysis under the unified mixed model of inheritance (major gene and multifactorial) is performed on families ascertained through 23 probands with hypoalphalipoproteinemia (depressed HDL-cholesterol, denoted HDL-c). Evidence for segregation of a recessive major gene for depressed HDL-c with frequency q = 0.116, in addition to multifactorial transmission (H = 0.572), is found in these families. Reanalysis of a subset of families with severely depressed HDL-c confirms the conclusions based on the original analysis, except that different definitions of "affection" give rise to different estimates of gene frequency. Our finding of a recessive mode of inheritance differs from previous claims for a dominant gene because previous analyses did not use a mixed model for segregation analysis of hypoalphalipoproteinemia. When the significant multifactorial background is neglected, we also find evidence for the invalid claim of a dominant gene. This demonstrates the necessity of using mixed models for determining the mode of inheritance of a given phenotype.

Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2.

Clinical symptoms, lipoprotein patterns, and apoE phenotypes were determined in 17 individuals with type III hyperlipoproteinemia (type III HLP) and in their relatives and spouses. The apoE phenotype E2/2 occurred in 15 type III HLP probands (88%) and the apoE phenotype E4/2 was found in 2 probands. In each of the families studied, the apoE phenotype inheritance was compatible with a model we previously proposed in which apoE is determined at a single genetic locus with three common alleles. The apoE phenotypes E4/4, E3/3, and E2/2 represent homozygosity for the apoE alleles epsilon4, epsilon3, and epsilon2, respectively, whereas the apoE phenotypes E4/3, E3/2, and E4/2 represent heterozygosity for the apoE alleles epsilon4/epsilon3, epsilon3/epsilon2, and epsilon4/epsilon2, respectively. Plasma lipids in 69 relatives of type III HLP probands were analyzed by apoE phenotype and revealed no significant differences between phenotypes in the levels of cholesterol, triglyceride, or HDL cholesterol. However, there were differences between the apoE phenotypes in LDL cholesterol levels (P = 0.01) and in the ratio of VLDL cholesterol/total triglyceride (ratio) (P < 0.01). Relatives with the apoE phenotype E2/2 had the lowest LDL cholesterol levels and the highest ratios. Of these eleven individuals with the apoE phenotype E2/2 who were not type III HLP probands, two males were taking lipid-lowering drugs, one male had mild angina at age 59, five individuals had ratios >0.25 and two had ratios >0.30 with the ratios for males (0.28 +/- 0.06) significantly greater than the ratios for females (0.17 +/- 0.06) (P < 0.01), and seven had evidence of floating betaVLDL on lipoprotein electrophoresis. In addition, when compared to a control group in the general population, the whole group of relatives had normal cholesterol and HDL cholesterol levels, slightly low LDL cholesterol levels, and almost twice elevated triglyceride levels. In summary, a) a very strong but not invariate association exists between type III HLP and the apoE phenotype E2/2 with some type III HLP individuals having the apoE phenotype E4/2; b) apoE phenotype inheritance is determined by three alleles at a single genetic locus; c) relatives of type III HLP probands, no matter what their apoE phenotype, have on the average nearly twofold elevated plasma triglyceride levels compared to a control population; and d) non-proband type III HLP individuals with the apoE phenotype E2/2 have been identified. As a group these individuals, particularly the males, show a tendency to express type III HLP, but clearly genetic or environmental factors other than the apoE phenotype E2/2 are required for the full phenotypic expression of this disease.-Breslow, J. L., V. I. Zannis, T. R. SanGiacomo, J. L. H. C. Third, T. Tracy, and C. J. Glueck. Studies of familial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2.