Analyses of toxic metals and essential minerals in the hair of Arizona children with autism and associated conditions, and their mothers.

The objective of this study was to assess the levels of 39 toxic metals and essential minerals in hair samples of children with autism spectrum disorders and their mothers compared to controls. Inductively coupled plasma-mass spectrometry was used to analyze the elemental content of the hair of children with autism spectrum disorders (n=51), a subset of their mothers (n=29), neurotypical children (n=40), and a subset of their mothers (n=25). All participants were recruited from Arizona. Iodine levels were 45% lower in the children with autism (p=0.005). Autistic children with pica had a 38% lower level of chromium (p=0.002). Autistic children with low muscle tone had very low levels of potassium (-66%, p=0.01) and high zinc (31%, p=0.01). The mothers of young children with autism had especially low levels of lithium (56% lower, p=0.005), and the young children (ages 3-6 yr) with autism also had low lithium (-30%, p=0.04). Low iodine levels are consistent with previous reports of abnormal thyroid function, which likely affected development of speech and cognitive skills. Low lithium in the mothers likely caused low levels of lithium in the young children, which could have affected their neurological and immunological development. Further investigations of iodine, lithium, and other elements are warranted.

Trace element analysis in hair: factors determining accuracy, precision, and reliability.

Trace element analysis in biological samples has improved significantly over the last 40 years. Improvements in instrumentation such as inductively coupled plasma-mass spectrometry and microwave digestion have resulted in improved precision, accuracy, reliability, and detection limits. The analysis of human scalp hair has benefited significantly from these improvements. A recent article in the Journal of the American Medical Association found significant inter-laboratory variation amongst several laboratories performing trace metal hair testing. It concluded that standardization was necessary to improve inter-laboratory comparability, and an accompanying commentary described the characteristics of a laboratory that should be used in performing hair analysis. The objective of this study is to demonstrate that good laboratory practices will generate precise, accurate, and reliable results. A method for establishing reference ranges and specific data on an analytical method will also be presented. The use of prescribed clinical quality control, including method validation, proficiency testing, split sampling, and good laboratory practices clearly demonstrates that measuring trace elements in hair can be analytically valid.

Cysteine metabolism and metal toxicity.

Chronic, low level exposure to toxic metals is an increasing global problem. The symptoms associated with the slow accumulation of toxic metals are multiple and rather nondescript, and overt expression of toxic effects may not appear until later in life. The sulfhydryl-reactive metals (mercury, cadmium, lead, arsenic) are particularly insidious and can affect a vast array of biochemical and nutritional processes. The primary mechanisms by which the sulfhydryl-reactive metals elicit their toxic effects are summarized. The pro-oxidative effects of the metals are compounded by the fact that the metals also inhibit antioxidative enzymes and deplete intracellular glutathione. The metals also have the potential to disrupt the metabolism and biological activities of many proteins due to their high affinity for free sulfhydryl groups. Cysteine has a pivotal role in inducible, endogenous detoxication mechanisms in the body, and metal exposure taxes cysteine status. The protective effects of glutathione and the metallothioneins are discussed in detail. Basic research pertaining to the transport of toxic metals into the brain is summarized, and a case is made for the use of hydrolyzed whey protein to support metal detoxification and neurological function. Metal exposure also affects essential element status, which can further decrease antioxidation and detoxification processes. Early detection and treatment of metal burden is important for successful detoxification, and optimization of nutritional status is paramount to the prevention and treatment of metal toxicity.

Preparation of hair for measurement of elements by inductively coupled plasma-mass spectrometry (ICP-MS).

The preparation of hair for the determination of elements is a critical component of the analysis procedure. Open-beaker, closed-vessel microwave, and flowthrough microwave digestion are methods that have been used for sample preparation and are discussed. A new digestion method for use with inductively coupled plasma-mass spectrometry (ICP-MS) has been developed. The method uses 0.2 g of hair and 3 mL of concentrated nitric acid in an atmospheric pressure-low-temperature microwave digestion (APLTMD) system. This preparation method is useful in handling a large numbers of samples per day and may be adapted to hair sample weights ranging from 0.08 to 0.3 g. After digestion, samples are analyzed by ICP-MS to determine the concentration of Li, Be, B, Na, Mg, Al, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Mo, Pd, Ag, Cd, Sn, Sb, I, Cs, Ba, Pt, Au, Hg, Tl, Pb, Bi, Th, and U. Benefits of the APLTMD include reduced contamination and sample handling, and increased precision, reliability, and sample throughput.

Determination of reference ranges for elements in human scalp hair.

Expected values, reference ranges, or reference limits are necessary to enable clinicians to apply analytical chemical data in the delivery of health care. Determination of references ranges is not straightforward in terms of either selecting a reference population or performing statistical analysis. In light of logistical, scientific, and economic obstacles, it is understandable that clinical laboratories often combine approaches in developing health associated reference values. A laboratory may choose to: 1. Validate either reference ranges of other laboratories or published data from clinical research or both, through comparison of patients test data. 2. Base the laboratory's reference values on statistical analysis of results from specimens assayed by the clinical reference laboratory itself. 3. Adopt standards or recommendations of regulatory agencies and governmental bodies. 4. Initiate population studies to validate transferred reference ranges or to determine them anew. Effects of external contamination and anecdotal information from clinicians may be considered. The clinical utility of hair analysis is well accepted for some elements. For others, it remains in the realm of clinical investigation. This article elucidates an approach for establishment of reference ranges for elements in human scalp hair. Observed levels of analytes from hair specimens from both our laboratory's total patient population and from a physician-defined healthy American population have been evaluated. Examination of levels of elements often associated with toxicity serves to exemplify the process of determining reference ranges in hair. In addition the approach serves as a model for setting reference ranges for analytes in a variety of matrices.

Effects of hyperlipidemias in hamsters on lipid transfer protein activity and unidirectional cholesteryl ester transfer in plasma.

Experiments were performed to characterize plasma lipid transfer protein activity (LTA), and the rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma of hamsters. Compared to rabbits, hamster plasma has about one-tenth the level of d greater than 1.21 LTA but a relatively high level of VLDL-triacylglycerols, and a higher fractional rate of HDL-[3H]CE transfer in plasma (in vitro) than predicted by the d greater than 1.21 LTA. Like the rat, hamster plasma contains an inhibitor(s) of LTA; the level of the inhibitor activity in d greater than 1.21 g/ml plasma was similar in normal and hyperlipoproteinemic hamsters. Hypertriglyceridemia in sucrose-fed hamsters did not affect LTA, cholesteryl ester transfer or the plasma level of HDL-CE. However, a comparable degree of hypercholesterolemia was associated with a 122% increase in plasma d greater than 1.21 LTA and a 63% increase in the fractional rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma. Cholesterol feeding in hamsters was associated with increased plasma levels of LDL-cholesterol and, to a lesser extent, with VLDL- and IDL-cholesterol.

High density lipoprotein metabolism in a rabbit model of hyperalphalipoproteinemia.

The potential utility of an animal model of hyperalphalipoproteinemia for examining the role of high density lipoprotein (HDL) in atherogenesis prompted the current studies. Preliminary data indicated that in rabbits high-coconut oil feeding for 30 days doubled plasma HDL-cholesterol levels, but did not affect lower density lipoproteins (LDL) (d less than 1.063 g/ml). Experiments were performed to examine the composition of these HDL and to determine the mechanism for the diet-induced increase in plasma HDL. Rabbits were fed commercial chow or chow plus 14% (w/w) coconut oil and blood samples were collected 18 h after feeding. Compared to chow-fed rabbits, peak levels of HDL-cholesterol were attained within 2 weeks, and coconut oil feeding doubled the plasma levels of HDL-cholesterol, phospholipids and protein for up to 4 months without affecting HDL lipid and apoprotein composition. After 3 months the diet also increased VLDL- (107%) and LDL-cholesterol (40%) levels, but the absolute increases in each of these lipoprotein fractions was less than half of that of HDL. Isotope kinetic studies of 125I-HDL protein indicated a doubled rate of production of HDL and no change in the efficiency of removal of HDL from plasma. These studies demonstrate that in the rabbit high-coconut oil feeding doubles the rate of production and turnover of apparently normal HDL particles. It is proposed that such an animal model could be utilized to examine directly the role of HDL in atherogenesis.

Dissociation between cholesterol secretion and plasma lipid transfer activity in rabbits.

Human and rabbit plasma contains a lipid transfer protein that transfers cholesteryl esters and triglycerides among the plasma lipoproteins and may also have a role in the movement of lipids into and out of cells. Little is known about the regulation of the activity of the lipid transfer protein, but in the rabbit, hypercholesterolemia is associated with increased plasma lipid transfer activity (LTA). Perfused rabbit livers secrete LTA, and hepatic cholesterol secretion is increased in rabbits with diet-induced hypercholesterolemia. Thus, experiments were performed with rabbits to determine if LTA is regulated by a concerted hepatic secretion of lipoprotein protein cholesterol and LTA. Rabbits were fed chow or chow plus coconut oil (14% wt/wt), and plasma lipids, LTA, and the rate of secretion of cholesterol into plasma were determined. Coconut oil feeding increased plasma cholesterol by 68%, LTA by 42%, and hepatic cholesterol secretion by 69%. Mevinolin (75 mg/day), an inhibitor of cholesterol biosynthesis, lowered LTA and plasma cholesterol without affecting the rate of secretion of cholesterol into plasma. These studies provide further evidence that, in the rabbit, plasma cholesterol and LTA are closely related, and the association is not likely to be caused by a concerted hepatic secretion of cholesterol and LTA.

Plasma lipid transfer activity in rabbits: effects of dietary hyperlipidemias.

Studies were performed in the rabbit to investigate the relationship between plasma lipids and the cholesteryl ester/triglyceride transfer activity of lipoprotein-deficient plasma (d greater than 1.21). The time courses for diet-induced changes in plasma lipids and the lipid (cholesteryl ester/triglyceride) transfer activity of lipoprotein-deficient plasma (LTA) were determined in rabbits fed a variety of hyperlipidemic diets. LTA was not altered within 36 h after a single high-cholesterol/oil meal despite a near doubling of the concentration of plasma cholesterol. With high cholesterol and/or high-fat feeding, LTA increased and reached new steady state levels within about 10 days with little additional change for up to 87 days even when plasma cholesterol continued to increase. For all diets, the greatest increments in LTA occurred about 5 days after initiation of experimental feeding. A low-cholesterol (0.05-0.2%, w/w) diet and a cholesterol-free, high-coconut oil diet (14%, w/w) were associated with comparable increases in both plasma cholesterol and LTA. A marked, concomitant increase in plasma triglycerides was not associated with a further increase in LTA in hypercholesterolemic rabbits. These data indicate that in the rabbit LTA increases in a parabolic manner with moderate and gross, diet-induced increases in plasma cholesterol, and the time courses for the diet-induced changes in LTA are similar for a variety of hyperlipidemic diets.

Disappearance and effects of exogenous lipid transfer activity in rats.

These studies were performed to determine the role of plasma lipid transfer activity in the regulation of plasma total and lipoprotein cholesterol in vivo. Partially purified human lipid transfer activity was injected into rats at a level similar to that of normal rabbit plasma, d greater than 1.21. The disappearance of exogenous lipid transfer activity from rat plasma was biphasic, with a 70% loss within 6 h. The remaining 30% was lost with a half-time of about 14 h. In the rat, short-term exposure (6 h) to high levels of lipid transfer activity resulted in a net transfer of cholesteryl esters from high density to d less than 1.019 lipoproteins, without affecting plasma total cholesterol. However, the lipid transfer activity-induced changes in lipoprotein cholesterol were not evident after 24 h, despite the fact that the lipid transfer activity of rat plasma d greater than 1.21 was similar to that of human plasma d greater than 1.21 during the preceding 18 h.

Parallel changes in plasma cholesterol and lipid transfer activity in pregnant rabbits.

The relationship between the concentration of plasma cholesterol and the lipid transfer activity (LTA) of lipoprotein-deficient plasma (d greater than 1.21) was studied in two models of pregnancy in the rabbit. Plasma cholesterol and the protein-mediated transfer of cholesteryl ester and triglyceride were monitored throughout gestation, 48 hr after parturition, and during lactation in New Zealand white (NZW) and heterozygous WHHL rabbits. Lipoprotein cholesterol was determined prior to and 48 hr after parturition. For both NZW and heterozygous WHHL rabbits, the progressive hypocholesterolemia of gestation was associated with parallel changes in LTA. Similarly, the rapid postpartum increase in plasma cholesterol was paralleled by increased LTA for both strains. In relation to basal values, the relative changes in plasma cholesterol and LTA were virtually identical. These data provide further evidence that in the rabbit plasma cholesterol and LTA are closely related.

Effects of short-term aerobic conditioning and high cholesterol feeding on plasma total and lipoprotein cholesterol levels in sedentary young men.

The plasma total cholesterol (TC) and lipoprotein cholesterol concentrations of sedentary young men (n = 23) were determined during 4 wk of controlled feeding and 6 wk of supervised aerobic conditioning. Subjects were assigned to dietary treatments of 400 mg cholesterol per day (M) or 1400 mg cholesterol per day (H); both diets had a P/S ratio of about 0.6. Dietary groups M and H were subdivided into exercise (MX and HX) and sedentary (MS and HS) groups. Compared to the sedentary groups, MX and HX exhibited significant (p less than 0.01) improvements in cardiorespiratory fitness. After 2 and 4 wk of high cholesterol feeding, group HS exhibited significant (p less than 0.05) elevations in TC (+30 +/- 7 and +32 +/- 9 mg/dl) with nonsignificant increases in very low-density lipoprotein cholesterol and low-density lipoprotein cholesterol. Group HX exhibited consistent weekly increases in high-density lipoprotein cholesterol (HDL-C) (from 46 +/- 3 mg/dl, the base level, to 53 +/- 4 mg/dl at wk 4) with aerobic conditioning. By combining exercise and sedentary group data at each level of dietary cholesterol it was shown that TC and HDL-C levels significantly (p less than 0.05) increased by the 4th wk of high cholesterol feeding. The TC/HDL-C ratio significantly (p less than 0.05) increased for the sedentary subjects as compared to all the exercising subjects by wk 4 of controlled feeding.

The influence of starvation and refeeding on the lipoprotein lipase activity of skeletal muscle and adipose tissue of lean and obese Zucker rats.

Lipoprotein lipase (LPL) activities of skeletal muscles, heart and adipose tissue were investigated during feeding, prolonged food restriction, and refeeding. The influence of the duration of starvation on adipose tissue LPL activity was to cause it to decrease throughout starvation, whereas heart LPL activity increased during the first 24 hours of fasting and then declined for the remainder of the fast. Starvation of 10-week-old female, lean and obese rats to 80% of initial body weight required 5 and 9 days, respectively. In fed controls, no differences between phenotypes were found for any tissue in the LPL activities expressed per gram tissue. However, obese rats exhibited significantly smaller muscle mass and a resulting 29% lower total skeletal muscle LPL activity. No phenotype differences were detected for tissue LPL activities during starvation or refeeding. During caloric restriction, the LPL activities were reduced in heart (-18%) and adipose (-52%) tissues, but skeletal muscle was unchanged except for the slow-twitch, oxidative soleus muscle, which was increased approximately two-fold. After refeeding to initial body weight, the LPL activity of heart returned to normal, but adipose tissue was dramatically increased (+300%) for both lean and obese Zucker rats. These data suggest that reduced skeletal muscle mass with normal LPL activity per gram tissue may contribute to an increased availability of plasma triglyceride fatty acids to adipose tissue of the genetically obese rat.