Zinc protoporphyrin: A metabolite with a mission.

Zinc protoporphyrin (ZnPP) is a normal metabolite that is formed in trace amounts during heme biosynthesis. The final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin. During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation. Evidence suggests that this metal substitution is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes. Because this zinc-for-iron substitution occurs predominantly within the bone marrow, the ZnPP/heme ratio in erythrocytes reflects iron status in the bone marrow. In addition, ZnPP may regulate heme catabolism through competitive inhibition of heme oxygenase, the rate-limiting enzyme in the heme degradation pathway that produces bilirubin and carbon monoxide. Physiological roles, especially relating to carbon monoxide and possibly nitric oxide production, have been suggested for ZnPP. Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism. Diagnostic determinations are applicable in a variety of clinical settings, including pediatrics, obstetrics, and blood banking. ZnPP analytical methodologies for clinical studies are discussed. In addition to diagnostic tests and metabolic studies, ZnPP has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia. Biochemical research techniques, both in vivo and in vitro, are described for further studies into the role of ZnPP in metabolism and physiology.

Quantitative study of the variability of hepatic iron concentrations.

BACKGROUND: The hepatic iron concentration (HIC) is widely used in clinical practice and in research; however, data on the variability of HIC among biopsy sites are limited. One aim of the present study was to determine the variability of HIC within both healthy and cirrhotic livers. METHODS: Using colorimetric methods, we determined HIC in multiple large (microtome) and small (biopsy-sized) paraffin-embedded samples in 11 resected livers with end-stage cirrhosis. HIC was also measured in multiple fresh samples taken within 5 mm of each other ("local" samples) and taken at sites 3-5 cm apart ("remote" samples) from six livers with end-stage cirrhosis and two healthy autopsy livers. RESULTS: The within-organ SD of HIC was 13-1553 microg/g (CV, 3.6-55%) for microtome samples and 60-2851 microg/g (CV, 15-73%) for biopsy-sized samples. High variability of HIC was associated with mild to moderate iron overload, because the HIC SD increased with increasing mean HIC (P <0.002). Livers with mean HIC >1000 microg/g exhibited significant biological variability in HIC between sites separated by 3-5 cm (remote sites; P <0.05). The SD was larger for biopsy-sized samples than for microtome samples (P = 0.02). CONCLUSION: Ideally, multiple hepatic sites would be sampled to obtain a representative mean HIC.

Ascorbic acid clearance in diabetic nephropathy.

The incidence of cardiovascular disease is increased in diabetic nephropathy. Increased oxidative stress in diabetes is believed to play an important role in the pathogenesis of atherosclerosis in diabetes. Since antioxidant vitamins, such as ascorbic acid, often are reduced in diabetes, we hypothesized that the renal clearance of ascorbic acid is increased in patients with diabetic nephropathy. Thirty-seven subjects with diabetic nephropathy were studies: 18 had microalbuminuria (30-300 mg/day albuminuria); the remainder had clinical nephropathy (> 300 mg/day albuminuria). Indices of glycemic control (glucose, hemoglobin A1C) and renal function (albuminuria and creatinine clearance) were measured in addition to serum and urinary ascorbic acid levels. Results showed that subjects with clinical nephropathy had lower mean plasma ascorbic acid (p=0.0009) and higher renal clearance of ascorbic acid (p=0.005) than those with microalbuminuria. Bivariate analysis revealed an inverse correlation between creatinine clearance and AA clearance (r=-0.42, p=0.009). There was a significant linear association between the quantity of albuminuria and ascorbic acid clearance (r=0.49, p=0.002). Thus, patients with diabetic nephropathy have reduced ascorbic acid levels due to increased ascorbic acid clearance. The decrease in antioxidant defense that arises from the low levels of vitamin C may contribute to the increased cardiovascular morbidity and mortality observed in this population.

Delayed bottle weaning and iron deficiency in southeast Asian toddlers.

We undertook this study to determine if culturally influenced feeding practices are associated with iron deficiency in infants and toddlers from low-income families. We obtained a dietary survey, illness history, hematocrit, and zinc protoporphyrin-to-heme ratio (ZPP/H) from 35 Southeast Asian children and 73 children of other ethnicities between ages 5 and 30 months. We confirmed iron deficiency by serum ferritin measurement in children with ZPP/H > 80 mmol/mol or evaluated them after a 3-month iron treatment. Sixty percent of the Southeast Asian children had elevated ZPP/H ratios, compared with 14% of children of other ethnicities. Follow-up studies confirmed iron deficiency in 12 of 21 Southeast Asian children with elevated ZPP/H; 75% (eight) of those with confirmed iron deficiency were 24 to 30 months of age. We found that toddler feeding practices differ between Southeast Asians and other ethnic groups. All 17 Southeast Asian toddlers were still bottle fed at their second birthday, compared with 10 of 21 same-age children of other ethnicities. Persistence of bottle feeding after 2 years of age was highly associated with elevation of ZPP/H in Southeast Asian children but not in other children. Clinicians need to be aware of this problem and carefully monitor iron status in children not weaned from the baby bottle by age 2 years. Changes in education practices and policies are needed to prevent iron deficiency from the overintake of cow's milk that results from prolonged bottle feedings in this ethnic group.

Environmental chemical exposures and disturbances of heme synthesis.

Porphyrias are relatively uncommon inherited or acquired disorders in which clinical manifestations are attributable to a disturbance of heme synthesis (porphyrin metabolism), usually in association with endogenous or exogenous stressors. Porphyrias are characterized by elevations of heme precursors in blood, urine, and/or stool. A number of chemicals, particularly metals and halogenated hydrocarbons, induce disturbances of heme synthesis in experimental animals. Certain chemicals have also been linked to porphyria or porphyrinuria in humans, generally involving chronic industrial exposures or environmental exposures much higher than those usually encountered. A noteworthy example is the Turkish epidemic of porphyria cutanea tarda produced by accidental ingestion of wheat treated with the fungicide hexachlorobenzene. Measurements of excreted heme precursors have the potential to serve as biological markers for harmful but preclinical effects of certain chemical exposures; this potential warrants further research and applied field studies. It has been hypothesized that several otherwise unexplained chemical-associated illnesses, such as multiple chemical sensitivity syndrome, may represent mild chronic cases of porphyria or other acquired abnormalities in heme synthesis. This review concludes that, although it is reasonable to consider such hypotheses, there is currently no convincing evidence that these illnesses are mediated by a disturbance of heme synthesis; it is premature or unfounded to base clinical management on such explanations unless laboratory data are diagnostic for porphyria. This review discusses the limitations of laboratory measures of heme synthesis, and diagnostic guidelines are provided to assist in evaluating the symptomatic individual suspected of having a porphyria.

Ascorbic acid determination with an automated enzymatic procedure.

Given the widespread interest in antioxidant nutrients, we have developed a new procedure that will permit the automated determination of plasma ascorbic acid (AA) concentration with a Roche Fara centrifugal analyzer. After the deproteinization of plasma with metaphosphoric acid, AA is oxidized to dehydroascorbic acid by AA oxidase. The product is coupled to o-phenylenediamine to produce a chromophore for which the absorbance is measured at 340 nm. The procedure allows much faster throughput than conventional HPLC methods while yielding results that correlate well and provide improved precision. Linearity extends beyond the reference range of 26.1-84.6 micromol/L, and severe hemolysis is the only interference identified.

Elevated zinc protoporphyrin associated with thalassemia trait and hemoglobin E.

OBJECTIVE: Increased zinc protoporphyrin/heme (ZPP/H) ratio has been used in pediatrics to screen for iron deficiency and lead poisoning. This study was conducted to determine whether common hereditary hemoglobin disorders (alpha- and beta-thalassemia traits, hemoglobin E) found in U.S. minority groups are associated with an increase in the ZPP/H ratio in an iron-sufficient population. METHODS: The database was compiled from hemoglobinopathy screens performed between 1987 and 1993 at a regional referral laboratory in Washington State. ZPP/H ratio and hemoglobin type were obtained for 326 subjects between the ages of 15 and 49 years of age who were iron sufficient (serum ferritin levels > or = 50 micrograms/L). RESULTS: The mean ZPP/H ratio was significantly higher (p < 0.01) for subjects with beta-thalassemia trait (87 +/- 32 micromol/mol), (alpha-thalassemia trait (73 +/- 37 micromol/mol), and hemoglobin E disorders (73 +/- 24 micromol/mol) than for subjects with normal hemoglobin values (60 +/- 8 micromol/mol). Fifty-one percent of subjects with beta-thalassemia trait, 22% with hemoglobin E, and 20% with alpha-thalassemia trait had elevated ZPP/H ratios (> 80 micromol/mol), compared with only 1.5% with normal hemoglobin values. CONCLUSIONS: The ZPP/H ratio is elevated in common hereditary hemoglobin disorders that mimic the microcytic anemia of iron deficiency, even in individuals without associated nutritional iron deficiency. For children who are treated for presumed iron deficiency, failure of the ZPP/H ratio to return to normal after adequate iron treatment, especially if microcytosis persists, indicates that a hereditary hemoglobin disorder may be present.

Multicenter evaluation of automated immunoturbidimetric assays for measurement of apolipoproteins A-I and B in serum and plasma.

Results of a multicenter evaluation of automated assays for measurement of apolipoproteins (apo) A-I and B with the Paramax analytical system are reported. Apo A-I and apo B response surface models were used to optimize concentrations of critical assay variables. Overall imprecision for apo A-I controls at concentrations of 1.01-1.61 g/L was 3.7-6.6%; overall imprecision for apo B controls at 1.00-1.61 g/L was 2.8-6.9%. There was no interference in apo A-I measurements. Albumin concentrations > 59 g/L resulted in a negative interference, and collection in sodium heparin caused a positive interference in apo B results. Apo A-I and apo B assays demonstrated acceptable agreement with comparative methods, although the Paramax apo B assays had a negative bias with respect to comparison methods. In 116 healthy individuals, serum apo A-I ranged from 0.97 to 2.05 g/L and serum apo B ranged from 0.51 to 1.32 g/L.

Nutrition in the clinical laboratory.

Nutrition is a developing facet of patient care that can be enhanced by appropriate laboratory support. The importance of the laboratory is underscored by the growing interest and appreciation for marginal malnutrition that can only be diagnosed by biochemical means. Clinical laboratories have the opportunity to provide leadership in advancing nutrition to the forefront of medical care. The overall quality and cost-effectiveness of medical care can be improved through the development of reliable tests to detect malnutrition, abandonment of tests of little value, emphasizing those tests of merit, and persuading clinicians of the benefits of measuring nutritional analytes. Nutrition test panels are an effective way of assuring appropriate and comprehensive laboratory support of nutritional care.

Laboratory monitoring of nutritional status in burn patients.

Most nutrition laboratory testing relies on serum concentrations of ingested nutrients, their coenzymes, proteins, or lipids. Alternatively, functional tests measure a specific physiological process or biochemical reaction. We compared these two approaches to nutritional assessment in intensive-care burn patients, in whom the serum concentrations of transthyretin (prealbumin), albumin, transferrin, carotene, retinol, ascorbic acid, copper, cholesterol, iron, and calcium were all below established reference ranges. In contrast, serum triglyceride concentrations were often above the reference range. Functional tests for thiamin, riboflavin, pyridoxine, and iron (by zinc protoporphyrin/heme ratio) in these patients all showed normal values. Dietary intake, weight trends, and nitrogen balances all indicated that these patients' estimated caloric and protein needs had been met. These findings suggest that static measurements of serum concentrations may be unreliable indicators of nutritional status in burn patients.

Chemiluminescent measurement of total urinary nitrogen for accurate calculation of nitrogen balance.

This instrumental method for total urinary nitrogen (TUN) is based on the principle of gas-phase chemiluminescence. Results correlate well with measurements of TUN by the Kjeldahl method, which has long provided the means to calculate nitrogen balances for nutritional management. In recent years, because of speed and convenience of measurement, determination of urinary urea nitrogen (UUN) has been substituted for Kjeldahl TUN. However, in patients requiring aggressive nutritional support, the UUN may not be a valid indicator of total nitrogen excretion. We compared nitrogen balances calculated for patients, using both UUN and chemiluminescence TUN data. For both normal and hospitalized populations, nitrogen balance calculated from UUN data exceeded that calculated from TUN data. We show that use of UUN data in calculating nitrogen balance may result in an incorrect assessment of many patients as being in positive nitrogen balance. TUN determined by chemiluminescence evidently provides a simple means of calculating nitrogen balance more nearly accurately.

Laser photobioactivation mechanisms: in vitro studies using ascorbic acid uptake and hydroxyproline formation as biochemical markers of irradiation response.

Clinical investigations of laser photobioactivation, or biostimulation, might be differently designed and more fruitful if knowledge of basic biochemical mechanisms were better understood. In this investigation, biochemical events identified as responses to 904 nm irradiation included increased ascorbic acid uptake by fibroblasts. These cells also showed increased hydroxyproline formation, and this was increased several-fold by the addition of proline to the medium. Maximum biochemical responses were observed at a pulse frequency of 67 Hz and a pulse width of 150 nsec with an energy density of approximately 7 mJ/cm2 per exposure. Elements in the mitochondrial cytochrome system are proposed as the radiation absorbing chromophore(s). Hypothetically, the energy generated is linked to ascorbic acid uptake, which in turn stimulates collagen synthesis.

Serum levels of selenium and retinol and the subsequent risk of cancer.

A nested case-control study was conducted to assess the relation between serum levels of selenium and retinol and the subsequent risk of cancer. During the years 1972-1984, in northwest Washington State, 156 cases of cancer were identified among members of two employee cohorts from whom specimens had been previously obtained and stored. Two hundred eighty-seven controls were selected from these cohorts and matched to cases on the basis of employer, age, sex, race, and date of blood draw. Selenium and retinol levels were measured by neutron activation and high pressure liquid chromatography, respectively. Information on known cancer risk factors was collected by telephone interviews of subjects and next of kin. Levels of selenium and retinol were unassociated with the incidence of cancer of all sites combined, both overall and within subgroups defined by age, sex, levels of the other micronutrient, time between blood draw and diagnosis, smoking status, and family history of cancer. These findings suggest that neither serum levels of selenium nor those of retinol have an appreciable effect on the risk of cancer.