Salting Out-Assisted Liquid-Liquid Extraction for Liquid Chromatography-Tandem Mass Spectrometry Measurement of Tacrolimus, Sirolimus, Everolimus, and Cyclosporine a in Whole Blood.

BACKGROUND: Therapeutic drug monitoring of the immunosuppressants tacrolimus, sirolimus, everolimus, and cyclosporine A is effectively performed by analyzing whole-blood samples using liquid chromatography coupled with tandem mass spectrometry. Samples are usually prepared using simple protein precipitation (PPT) with methanol and zinc sulfate (ZnSO4). Significant sample dilution is necessary to obtain clean extracts but may increase the limit of quantification of the method. Salting out-assisted liquid-liquid extraction (SALLE) was explored as a novel sample preparation method for measuring these drugs in blood. METHOD: SALLE, which simply consists of LLE with a water-miscible solvent where phase separation is achieved by adding salt, was used to analyze treated blood samples. RESULTS: SALLE allowed direct injection of a 5-µL extract from the upper solvent phase into a reversed phase LC column, which would not be feasible using standard LLE. Compared with PPT, SALLE provided better extraction efficiencies and more ion enhancement, resulting in limit of quantification of 0.4, 1.4, 0.06, and 0.4 ng/mL for tacrolimus, sirolimus, everolimus, and cyclosporine A, respectively. Full-method validation was performed, including a comparison of results with those of another laboratory. A ≤10% bias was observed for tacrolimus and cyclosporine A, whereas further investigation of that for sirolimus (-12%) and everolimus (-18%) revealed that it was caused by the different calibrators used. CONCLUSIONS: This is the first report of the use of SALLE for the measurement of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood. The advantages of SALLE over PPT and conventional LLE would make it an attractive sample preparation method for clinical laboratories.

Short Term Oral Zinc Supplementation among Babies with Neonatal Sepsis for Reducing Mortality and Improving Outcome - A Double-Blind Randomized Controlled Trial.

OBJECTIVE: To evaluate the efficacy of short term zinc supplementation on the mortality rate and neurodevelopment outcome in neonates with sepsis at 12 mo corrected age. METHODS: The clinical trial was undertaken in the neonatal intensive care unit of JIPMER during the time period from September 2013 through December 2016. Neonates with clinical manifestations of sepsis who exhibited two positive screening tests (microESR, C- reactive protein, band cell count) were included and randomized into no zinc and zinc group. The intervention was zinc sulfate monohydrate given at a dose of 3 mg/kg twice a day orally for 10 d along with standard antibiotics. The no zinc group was on antibiotic treatment. Blood samples from both groups were collected at baseline and after day 10. Babies were carefully discharged from the hospital. The babies were followed up till 12 mo corrected age using DASII (Development Assessment Scale for Indian Infants). RESULTS: At the time of enrolment, patient characteristics were similar in both the groups. The mortality rate was significantly higher in no zinc compared to zinc group (5 vs. 13; P = 0.04). Although motor development quotient was similar, mental development quotient was significantly better among babies who received zinc supplementation. CONCLUSIONS: Short term zinc supplementation of newborns with sepsis reduces mortality and improves mental development quotient at 12 mo of age.

Effects of zinc and magnesium supplements on postpartum depression and anxiety: A randomized controlled clinical trial.

Postpartum anxiety and depression are prevalent disorders. The authors of this study aimed to determine the effects of zinc and magnesium supplements on depressive symptoms and anxiety in postpartum women referred to three governmental, educational hospitals in Tabriz, Iran during 2014-2015. In this triple-blind, randomized, controlled clinical trial, the participants were randomly assigned to the zinc sulfate, magnesium sulfate, and placebo groups (n = 33 per group). The intervention groups received a 27-mg zinc sulfate tablet or 320-mg magnesium sulfate tablet per day for 8 weeks, whereas the control group received a placebo tablet each day during the same period. The Edinburgh Postnatal Depression Scale and the Spielberger State-Trait Anxiety Inventory were completed before and 8 weeks after the intervention. Blood samples were drawn from each participant to determine serum levels of zinc and magnesium before intervention at 48 hours after delivery. Also, a 24-hour dietary questionnaire was used during the first and last 3 days of the intervention. Adjusting for baseline scores as well as zinc and magnesium serum levels, no significant difference was observed between groups 8 weeks after delivery in mean scores of depressive symptoms (p = .553), state anxiety (p = .995), and trait anxiety (p = .234). This study concluded magnesium and zinc did not reduce postpartum anxiety and depressive symptoms.

Subclinical zinc deficiency impairs pancreatic digestive enzyme activity and digestive capacity of weaned piglets.

This study investigated the effects of short-term subclinical Zn deficiency on exocrine pancreatic activity and changes in digestive capacity. A total of forty-eight weaned piglets were fed ad libitum a basal diet (maize and soyabean meal) with adequate Zn supply (88 mg Zn/kg diet) during a 2-week acclimatisation phase. Animals were then assigned to eight dietary treatment groups (n 6) according to a complete randomised block design considering litter, live weight and sex. All pigs were fed restrictively (450 g diet/d) the basal diet but with varying ZnSO4.7H2O additions, resulting in 28·1, 33·6, 38·8, 42·7, 47·5, 58·2, 67·8 and 88·0 mg Zn/kg diet for a total experimental period of 8 d. Pancreatic Zn concentrations and pancreatic activities of trypsin, chymotrypsin, carboxypeptidase A and B, elastase and α-amylase exhibited a broken-line response to stepwise reduction in dietary Zn by declining beneath thresholds of 39·0, 58·0, 58·0, 41·2, 47·5, 57·7 and 58·0 mg Zn/kg diet, respectively. Furthermore, carboxypeptidase B and α-amylase activities were significantly lower in samples with reduced pancreatic Zn contents. Coefficients of faecal digestibility of DM, crude protein, total lipids and crude ash responded similarly to pancreatic enzyme activities by declining below dietary thresholds of 54·7, 45·0, 46·9 and 58·2 mg Zn/kg diet, respectively. In conclusion, (1) subclinical Zn deficiency impaired pancreatic exocrine enzymes, (2) this response was connected to pancreatic Zn metabolism and (3) the decline in catalytic activity impaired faecal digestibility already after 1 week of insufficient alimentary Zn supply and very early before clinical deficiency symptoms arise.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 µM Zn), or with 15 or 50 µM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 µM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-ß/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

Bioavailability of zinc sources and their interaction with phytates in broilers and piglets.

Zinc (Zn) is essential for swine and poultry and native Zn concentrations in feedstuffs are too low to meet their Zn requirement. Dietary Zn bioavailability is affected by phytate, phytase and Zn supplemented in organic form is considered as more bioavailable than inorganic sources. A meta-analysis using GLM procedures was processed using broiler and piglet databases to investigate, within the physiological response of Zn, (1) the bioavailability of inorganic and organic Zn sources (Analysis I); (2) the bioavailability of native and inorganic Zn dependent from dietary phytates, vegetal and supplemental phytase activity (Analysis II). Analysis I: the bioavailability of organic Zn relative to inorganic Zn sources ranged, depending on the variable, from 85 to 117 never different from 100 (P > 0.05). The coefficients of determination of the regressions were 0.91 in broilers and above 0.89 in piglets. Analysis II: in broilers, bone Zn was explained by supplemental Zn (linear and quadratic, P < 0.001) and by supplemental phytase (linear, P < 0.001). In piglets, the interaction between dietary Zn and phytates/phytases was investigated by means of a new variable combining dietary phytic phosphorus (PP) and phytase activity. This new variable represents the remaining dietary PP after its hydrolysis in the digestive tract, mainly due to phytase and is called non-hydrolyzed phytic phosphorus (PP(NH)). Bone Zn was increased with native Zn (P < 0.001), but to a lower extent in high PP or low phytase diets (ZN(N) × PP(NH), P < 0.001). In contrast, the increase in bone zinc in response to supplemental Zn (P < 0.001) was not modulated by PP(NH) (P > 0.05). The coefficients of determination of the regressions were 0.92 in broilers and above 0.92 in piglets. The results from the two meta-analyses suggest that (1) broilers and piglets use supplemented Zn, independent from Zn source; (2) broiler use native Zn and the use is slightly enhanced with supplemental phytase; (3) however, piglets are limited in the use of native Zn because of the antagonism of non-hydrolyzed dietary phytate. This explains the higher efficacy of phytase in improving Zn availability in this specie.

Preventive effects of selenium yeast, chromium picolinate, zinc sulfate and their combination on oxidative stress, inflammation, impaired angiogenesis and atherogenesis in myocardial infarction in rats.

PURPOSE: Accumulating evidences suggest a critical role of trace metal dyshemostasis in oxidative stress and cardiac dysfunction after myocardial infarction (MI). This study investigated the cardioprotective effects of selenium yeast (Se), chromium picolinate Cr(pic)3, zinc sulfate (Zn) and their combination on isoproterenol (ISO)-induced MI. METHODS: Rats were divided into six groups: normal control, ISO control, Se-pretreated (0.1 mg/kg), Cr(pic)3-pretreated (400 µg/kg), Zn-pretreated (30 mg/kg) and metal combination-pretreated groups. All metals were administered for 28 days and at the 27th day, MI was induced by subcutaneous injection of ISO (85 mg/kg) once for two consecutive days. RESULTS: ISO control group showed hyperlipidemia, elevation of cardiac biomarkers and lipid peroxidation and increased immunostaining of p47 phox NADPH oxidase subunit in addition to decreased levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Cardiac levels of tumor necrosis factor-α (TNF-α) were increased, while vascular endothelial growth factor (VEGF, the major angiogenic factor) was decreased. Pretreatment with Se normalized the cardiac enzymes, lipid peroxidation, GSH, SOD, CAT, GPx, TNF-α and VEGF (P<0.001) and reduced the immunostaining of p47 phox subunit. However, Se failed to correct the dyslipidemia. Cr(pic)3 significantly improved lipid profile (P<0.001) and all other biochemical deviations except for VEGF. Zn, but to lesser extent, reduced the oxidative damage and TNF-α levels and improved both dyslipidemia and angiogenesis. Combination therapy exhibited less prominent protection compared to individual metals. CONCLUSION: Daily supplementation with trace metals is promising for improving myocardial performance via preventing oxidative damage, induction of angiogenesis, anti-inflammatory and/or anti-hyperlipidemic mechanisms.

The disposition and intestinal absorption of zinc in rats.

The variety of physiologic and biologic functions of zinc is expected to enable the development of zinc-related medicines. In this study, the distribution of endogenous zinc, the disposition after intravenous injection, and the intestinal absorption of zinc were investigated in vivo using rats from the viewpoints of pharmaceutical science and pharmacokinetics. High levels of endogenous zinc were observed in bone, testis, and liver. RT-PCR analysis on the mRNA of metallothionein in tissues clarified that it is significantly correlated with the distribution of zinc, suggesting that zinc is accumulated in tissues as a complex with MT. Following intravenous injection, uptake of zinc was high in liver, spleen, pancreas, kidney, and intestine. Fractional absorptions of zinc after oral administration to fasted rats were greater than those to fed rats, suggesting that some factors in diet inhibit the absorption of zinc. In fasted rats, fractional absorption was slightly decreased in high-dose group, suggesting the involvement of carrier-mediated transport. Study utilizing an in situ closed-loop method also indicated saturable intestinal absorption of zinc. These findings will further the research and development of zinc-related medicines by providing basic and important information on zinc.

Exogenous zinc improves blood fluidity but has no effect on the mechanisms of vascular response to acetylcholine iontophoresis in humans.

Recent findings in cellular signaling function of zinc through the mobilization intracellular calcium or by inducing ATP release suggest that extracellular zinc plays an important role in many physiological functions. However, such an extracellular signaling action of zinc for most cells is not known. Therefore, we investigated whether zinc plays any role in endothelium- dependent acetylcholine (ACh)-induced vasodilatation in microvascular beds. Transdermal iontophoresis was used to transport ACh through the forearm skin and cutaneous perfusion was measured using a laser Doppler flowmeter (LDF). Experiments were repeated using (1) zinc instead of ACh to test the effect of zinc ions alone and (2) concomitant iontophoresis of ACh and zinc to explore the effect of zinc on ACh-induced vasodilatation. Although zinc augments blood flow, curve-fitting to LDF signals indicate that zinc has no effect on the neural and endothelial component of ACh-induced vasodilatation. Additionally, no effect of Zn2+ on blood flow was found during its iontophoresis alone. Therefore, it is suggested from the Fourier analysis of LDF signals that the Zn+ might influence blood fluidity by its action on red blood cells deformability/ aggregability during a high-blood-flow condition, which might, in turn, decrease blood viscosity and improve blood flow in vivo.

Zinc L-carnosine protects against mucosal injury in portal hypertensive gastropathy through induction of heat shock protein 72.

BACKGROUND AND AIMS: Increased susceptibility to gastric mucosal injury is observed in portal hypertensive gastropathy (PHG). In this study, the effects of zinc L-carnosine, an anti-ulcer drug, were evaluated on expression of heat shock protein (hsp) 72 and cytoprotection in gastric mucosa in a rat model of PHG. METHODS: Portal hypertensive gastropathy with liver cirrhosis was induced by bile duct ligation for 4 weeks in male Sprague-Dawley rats. Expression of gastric mucosal hsp72 was evaluated by Western blotting at 6 h after intragastric administration of L-carnosine, zinc sulfate, or zinc L-carnosine. Blood was also collected for determination of serum zinc level. Mucosal protective abilities against hydrochloric acid (HCl) (0.6N) followed by pretreatment with L-carnosine, zinc sulfate or zinc L-carnosine were also studied. RESULTS: L-carnosine, zinc sulfate, and zinc L-carnosine induced hsp72 in gastric mucosa of rats with bile duct ligation. Zinc sulfate and zinc L-carnosine suppressed HCl-induced mucosal injury. However, L-carnosine could not suppress HCl-induced mucosal injury. Serum zinc levels were significantly elevated after zinc L-carnosine administration. Furthermore, pretreatment with zinc L-carnosine (30-300 mg/kg) increased the expression of hsp72 in gastric mucosa and prevented HCl-induced mucosal injury in rats with bile duct ligation in a dose-dependent manner. CONCLUSIONS: Zinc derivatives, especially zinc L-carnosine, protected portal hypertensive gastric mucosa with increased hsp72 expression in cirrhotic rats. It is postulated that zinc L-carnosine may be beneficial to the mucosal protection in PHG as a 'chaperone inducer'.

Efeito da suplementação de zinco a crianças de 1 a 5 anos de idade / Effects of zinc supplementation on 1- to 5-year old children

OBJETIVO: Avaliar o impacto da suplementação com zinco sobre os parâmetros nutricionais e bioquímicos entre crianças de 12 a 59 meses de idade. MÉTODOS: Foi realizado um estudo clínico randomizado unicego com 58 crianças entre 12 e 59 meses participantes do Programa Governamental de Combate a Carências Nutricionais, que fornecia mensalmente 2 kg de leite fortificado com ferro. O grupo intervenção (n = 28) foi suplementado com 10 mg/dia de sulfato de zinco por 4 meses, e o grupo controle (n = 30) recebeu solução placebo. Para avaliação do estado nutricional, utilizaram-se os indicadores peso por estatura e estatura por idade, expressos em escores z, do padrão de referência NCHS (National Center for Health Statistics), parâmetros bioquímicos de ferro e zinco séricos e concentração de hemoglobina e hematócrito. RESULTADOS: A suplementação com zinco não interferiu significativamente sobre as condições antropométricas das crianças. Ambos os grupos apresentavam concentrações iniciais baixas de zinco sérico. Após o término do período de intervenção, a variação nos níveis médios de hemoglobina (p = 0,002) e as concentrações de hematócrito (p = 0,001), zinco (p = 0,023) e ferro séricos (p = 0,013) foram significativamente mais elevadas no grupo suplementado. CONCLUSÃO: A suplementação com zinco promoveu melhora na resposta hemoglobínica e normalizou a concentração sérica de zinco. Os resultados mostram a importância de se estabelecer políticas de combate a carências nutricionais que também possam dar atenção à carência de zinco.

The effect of zinc supplementation on linear growth, body composition, and growth factors in preterm infants.

OBJECTIVE: The aim of our study was to evaluate the effect of zinc supplementation on linear growth, body composition, and growth factors in premature infants. DESIGN: Thirty-six preterm infants (gestational age: 32.0 +/- 2.1 weeks, birth weight: 1704 +/- 364 g) participated in a longitudinal double-blind, randomized clinical trial. They were randomly allocated either to the supplemental (S) group fed with a standard term formula supplemented with zinc (final content 10 mg/L) and a small quantity of copper (final content 0.6 mg/L), or to the placebo group fed with the same formula without supplementation (final content of zinc: 5 mg/L and copper: 0.4 mg/L), from 36 weeks postconceptional age until 6 months corrected postnatal age. At each evaluation, anthropometric variables and bioelectrical impedance were measured, a 3-day dietary record was collected, and a blood sample was taken. We analyzed serum levels of total alkaline phosphatase, skeletal alkaline phosphatase (sALP), insulin growth factor (IGF)-I, IGF binding protein-3, IGF binding protein-1, zinc and copper, and the concentrations of zinc in erythrocytes. RESULTS: The S group had significantly higher zinc levels in serum and erythrocytes and lower serum copper levels with respect to the placebo group. We found that the S group had a greater linear growth (from baseline to 3 months corrected age: Delta score deviation standard length: 1.32 +/-.8 vs.38 +/-.8). The increase in total body water and in serum levels of sALP was also significantly higher in the S group (total body water: 3 months; corrected age: 3.8 +/-.5 vs 3.5 +/-.4 kg, 6 months; corrected age: 4.5 +/-.5 vs 4.2 +/-.4 kg; sALP: 3 months; corrected age: 140.2 +/- 28.7 vs 118.7 +/- 18.8 micro g/L). CONCLUSIONS: Zinc supplementation has a positive effect on linear growth in premature infants.

Zinc supplementation decreases total thyroid hormone concentration in small ruminants.

The effect of dietary zinc (Zn) supplementation on plasma Zn and serum thyroid hormones was evaluated in healthy male Merino lambs and Angora goats. A total of 12 lambs and 12 goats were divided into two equal groups as control and Zn groups in separate experiments. The lambs and goats of the control groups were fed basal rations alone. The Zn contents of these rations prepared for lambs and goats were 40 mg/kg and 35 mg/kg in dry matter (DM), respectively. Both species of animals in the Zn groups were fed a basal ration supplemented with zinc sulphate adjusted to 250 mg Zn/kg diet in DM. The feeding trial lasted for 12 weeks in lambs and 8 weeks in goats. Blood samples were taken from the jugular vein at 4-week intervals. Both animal species in the Zn groups had higher plasma Zn values than the controls throughout the experimental period, except in the 4th week in goats. However, the levels of serum total thyroxine (T4) and triiodothyronine (T3) were lower in the lambs and goats of the Zn groups, except in the 4th week, as compared to those in the controls. Moreover, serum total thyroid hormone levels of the goats were higher at the 4th week than at the 8th week. Although there was a decrease in the levels of free thyroxine and triiodothyronine of both small ruminant species in the Zn groups when compared to the controls, these alterations were not statistically significant. These results may show that zinc supplementation to the diet at this dose reduces total thyroid hormone levels in small ruminants but does not yet impair the euthyroid status of the organism.

Effects of folic acid and zinc sulfate on male factor subfertility: a double-blind, randomized, placebo-controlled trial.

OBJECTIVE: To study the effects of folic acid and zinc sulfate treatment on semen variables in fertile and subfertile men. DESIGN: Double-blind, placebo-controlled interventional study. SETTING: Two outpatient fertility clinics and nine midwifery practices in The Netherlands. PARTICIPANT(S): One hundred eight fertile and 103 subfertile men. INTERVENTION(S): Both groups were randomly assigned to receive one of four treatments for 26 weeks: folic acid and placebo, zinc sulfate and placebo, zinc sulfate and folic acid, and two placebos. Folic acid was given at a daily dose of 5 mg, and zinc sulfate was given at a daily dose of 66 mg. MAIN OUTCOME MEASURE(S): Before and after treatment, standardized semen and blood samples were obtained for determinations of sperm concentration, motility, and morphology according to World Health Organization guidelines; semen morphology according to strict criteria; and blood folate and zinc concentrations. Effects of the four interventions were evaluated separately in subfertile and fertile men. RESULT(S): Subfertile men demonstrated a significant 74% increase in total normal sperm count and a minor increase of 4% abnormal spermatozoa. A similar trend was observed in fertile men. Pre-intervention concentrations of folate and zinc in blood and seminal plasma did not significantly differ between fertile and subfertile men. CONCLUSION(S): Total normal sperm count increases after combined zinc sulfate and folic acid treatment in both subfertile and fertile men. Although the beneficial effect on fertility remains to be established, this finding opens avenues of future fertility research and treatment and may affect public health.

In vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using Comet assay.

Single stranded DNA breaks induced by Zinc sulfate in mice has been studied in vivo using Alkaline Single Cell Gel Electrophoresis (Comet assay). Mice were administered orally with doses of 5.70, 8.55, 11.40, 14.25, 17.10 and 19.95 mg/kg body weight of zinc sulfate respectively. The samples of whole blood were collected at 24, 48, 72, 96 hr and first week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-lengths. Results indicated a significant DNA damage at all the doses after treatment with zinc sulfate when compared to controls showing a clear dose-dependent response (p < 0.05). A gradual decrease in the tail-lengths from 48 hr post-treatment onwards was observed indicating a time dependent decrease in the DNA damage. The study confirms that zinc sulfate causes significant DNA damage at the doses used as revealed by comet assay.

Blackcurrant seed oil, zinc, and fetal alcohol syndrome.

The effects of dietary supplementation by blackcurrant seed oil (BSO) and zinc sulphate on PGI2 gastric release and on serum zinc levels were investigated in 50 pregnant Sprague-Dawley rats which received watery alcohol solutions from 5% to 30% (v/v) as well as in their newborns. Blood zinc was assayed by atomic absorption spectroscopy; PGI2 was evaluated by platelet aggregation PGI2-dependent test. Dietary supplementation by BSO increased gastric release of PGI2 both in the mothers and their newborns; blood zinc levels were significantly (p < 0.01) lower in both vs control. The content of PGI2 in the stomachs was significantly (p < 0.01) higher in the mothers treated by zinc sulphate vs their new-borns. Protection of gastric mucosa of the new-born rats from alcohol fetal exposure might depend on polyunsaturated fatty acids (PUFA) dietary supplementation, because of the capability of PUFA to cross placenta; the efficacy in the protection might be monitored by platelet aggregation PGI2-dependent test.

Lack of effect of zinc supplementation on antipyrine clearance in alcoholic liver disease.

METHODS: The effect of zinc supplementation on antipyrine clearance was evaluated in 14 outpatients with stable alcoholic liver disease, of whom nine had biopsy proven alcoholic cirrhosis. RESULTS: There was no change in antipyrine clearance after 14 days of zinc supplementation (median 12.5 vs 12.9 ml.min-1). However, a significant increase in P-prothrombin-proconvertin was found. There was a positive correlation between S-zinc and antipyrine clearance at inclusion (rs = 0.76) as well as after zinc supplementation (rs = 0.72). CONCLUSION: No effect of zinc supplementation on antipyrine clearance was found. The positive correlation between S-zinc and antipyrine clearance could be due to the confounding effect of alcoholic liver disease.